一種抗胎盤樣硫酸軟骨素的嵌合抗原受體及其應用的制作方法

文檔序號：12284572閱讀：399來源：國知局

本發(fā)明涉及腫瘤的細胞免疫治療領(lǐng)域，尤其涉及一種抗胎盤樣硫酸軟骨素的嵌合抗原受體及其應用，具體為以腫瘤特異靶點pl-CS為基礎的嵌合抗原受體T(CAR-T)細胞技術(shù)的構(gòu)建方法及其在抗腫瘤治療中的應用。

背景技術(shù)：

嵌合抗原受體T細胞(CAR-T)技術(shù)是目前在進行大規(guī)模臨床試驗的新型細胞療法。在急性白血病和非霍奇金淋巴瘤的治療上有著顯著的療效，被認為是最有前景的腫瘤治療方式之一。其基本技術(shù)是通過基因工程手段，將T細胞改造成不依賴HLA的方式識別腫瘤抗原，使得CAR-T細胞相較于天然T細胞具有更強的識別和殺傷腫瘤細胞的能力。CAR-T的核心部件是CAR，CAR一般包括一個腫瘤相關(guān)抗原結(jié)合區(qū)，通常來源于腫瘤相關(guān)抗原的單克隆抗體scFV段；一個胞外鉸鏈區(qū)；一個跨膜區(qū)和一個胞內(nèi)刺激信號區(qū)。

CAR-T技術(shù)成功的關(guān)鍵是腫瘤抗原即靶點的選擇。理想的抗原是在腫瘤細胞表面高表達，在正常細胞不表達的抗原。只有選擇這樣的抗原，才會使CAR-T細胞專一的結(jié)合并殺傷腫瘤細胞，而對正常細胞不結(jié)合，不會對正常組織造成傷害。但這樣理想的抗原并不多，大多數(shù)抗原是在腫瘤組織中高表達或者過度表達，在正常組織中低表達。目前，CAR-T在實體腫瘤的治療遠不及白血病的治療效果，其原因之一就是各實體瘤的中均沒有找到非常理想的靶標。

CAR-T技術(shù)用于治療實體瘤的效果不好的另一個原因是腫瘤免疫抑制微環(huán)境的存在。腫瘤免疫抑制微環(huán)境是腫瘤發(fā)生發(fā)展過程中形成的內(nèi)環(huán)境，由腫瘤細胞，間質(zhì)細胞，免疫調(diào)節(jié)細胞，微血管，組織液，細胞因子，細胞外基質(zhì)等組成。由于免疫微環(huán)境的存在，免疫殺傷細胞無法有效到達腫瘤細胞部位或者到達目標部位后無法對腫瘤細胞進行有效的殺傷。CAR-T細胞要對實體瘤進行有效的治療，必需突破腫瘤免疫微環(huán)境限制，進入實體瘤實現(xiàn)對腫瘤細胞的殺傷。

CN 104788573 A公開了一種嵌合抗原受體hCD19scFv-CD8α-CD28-CD3ζ及其用途，該嵌合抗原受體由抗人CD19單克隆抗體HI19a輕鏈和重鏈可變區(qū)(hCD19scFv)、人CD8α鉸鏈區(qū)、人CD28跨膜區(qū)和胞內(nèi)區(qū)、以及人CD3ζ胞內(nèi)區(qū)結(jié)構(gòu)串聯(lián)構(gòu)成。該嵌合抗原受體用于修飾T淋巴細胞，修飾后的T細胞(CAR-T細胞)能用于表面CD19陽性的腫瘤的治療。該專利的嵌合抗原受體只針對于表面CD19陽性的腫瘤，且識別效果不好，因此，篩選合適的腫瘤靶標，同時還能突破腫瘤微環(huán)境，是CAR-T技術(shù)治療實體瘤的關(guān)鍵。

硫酸軟骨素(CS)是共價連接在蛋白質(zhì)上形成蛋白聚糖的一類糖胺聚糖。硫酸軟骨素廣泛分布于動物組織的細胞外基質(zhì)和細胞表面，糖鏈由交替的葡萄糖醛酸和N-乙酰半乳糖胺(又稱N-乙酰氨基半乳糖)二糖單位組成，通過一個似糖鏈接區(qū)連接到核心蛋白的絲氨酸殘基上。硫酸軟骨素存在于從線蟲到人除植物外的所有生物中，發(fā)揮著許多重要的生理功能。雖然多糖的主鏈結(jié)構(gòu)并不復雜，但就硫酸化程度、硫酸基和兩種差異向異構(gòu)糖醛酸在鏈內(nèi)的分布來說，呈現(xiàn)高度的不均一性。硫酸軟骨素的精細結(jié)構(gòu)決定著功能的特異性和與多種蛋白質(zhì)分子的相互作用。

胎盤中存在著一種特殊的硫酸軟骨素，其糖基化模式與常規(guī)的硫酸軟骨素不同，瘧原蟲中存在一種名為VAR2CSA蛋白，可以特異的結(jié)合胎盤中的硫酸軟骨素，而在腫瘤細胞的表面，存在著胎盤樣硫酸軟骨素(pl-CS)，因此，pl-CS是CAR-T技術(shù)治療腫瘤的理想靶點。

技術(shù)實現(xiàn)要素：

針對目前CAR-T技術(shù)治療腫瘤中靶點選擇不十分理想，以及腫瘤微環(huán)境影響CAR-T技術(shù)治療效果的情況，本發(fā)明提供一種抗胎盤樣硫酸軟骨素的嵌合抗原受體及其應用，所述嵌合抗原受體，可以更特異的識別腫瘤細胞，對腫瘤微環(huán)境具有更好的靶向性。

為達此目的，本發(fā)明采用以下技術(shù)方案：

一方面，本發(fā)明提供一種抗胎盤樣硫酸軟骨素的嵌合抗原受體，所述嵌合抗原受體主要包括抗pl-CS的抗原識別區(qū)、鉸鏈區(qū)、跨膜區(qū)和胞內(nèi)區(qū)串聯(lián)構(gòu)成；

其中，所述抗pl-CS的抗原識別區(qū)為瘧原蟲蛋白VAR2CSA、瘧原蟲蛋白VAR2CSA的部分肽段或pl-CS抗體中的任意一種；

所述瘧原蟲蛋白VAR2CSA的部分肽段為瘧原蟲蛋白VAR2CSA中的氨基酸數(shù)量大于500的肽段。

本發(fā)明中，所述瘧原蟲蛋白VAR2CSA的部分肽段指的是瘧原蟲蛋白VAR2CSA中的氨基酸數(shù)量大于500的肽段，可以是瘧原蟲蛋白VAR2CSA中的任意500個氨基酸或是500個氨基酸以上的肽段都是可行的。本發(fā)明中，瘧原蟲蛋白VAR2CSA、瘧原蟲蛋白VAR2CSA的部分肽段ID1-ID2a和pl-CS抗體均可以特異性結(jié)合pl-CS，pl-CS幾乎存在于所有類型的腫瘤細胞表面，而正常細胞的表面不存在pl-CS；選擇pl-CS為腫瘤的特異性靶點，以VAR2CSA作為CAR的識別結(jié)構(gòu)域來識別腫瘤的pl-CS，通過VAR2CSA識別pl-CS的方式，可以使CAR-T細胞特異的識別腫瘤細胞，更容易靶向到腫瘤微環(huán)境，進入到腫瘤組織內(nèi)部，而不會對正常的細胞和組織造成傷害；此外，pl-CS還是腫瘤細胞外基質(zhì)的組成部分，是形成腫瘤微環(huán)境的物質(zhì)基礎，選擇pl-CS為靶點，有利于CAR-T細胞突破腫瘤免疫抑制微環(huán)境。

優(yōu)選地，所述瘧原蟲蛋白VAR2CSA為瘧原蟲pf 3D7的VAR2CSA蛋白和/或瘧原蟲pf FCR3的VAR2CSA蛋白。

優(yōu)選地，所述瘧原蟲pf 3D7的VAR2CSA蛋白的氨基酸序列為SEQ ID NO.3，核酸序列為SEQ ID NO.4。

優(yōu)選地，所述瘧原蟲pf FCR3的VAR2CSA蛋白的氨基酸序列為SEQ ID NO.5，核酸序列為SEQ ID NO.6。

優(yōu)選地，所述瘧原蟲蛋白VAR2CSA的部分肽段為ID1-ID2a，所述ID1-ID2a的氨基酸序列為SEQ ID NO.7，核酸序列為SEQ ID NO.8。

本發(fā)明中，所述ID1-ID2a是這個部分肽段中，長度比較小，是效果最好的一段。

優(yōu)選地，所述鉸鏈區(qū)為人CD8α鉸鏈區(qū)。

優(yōu)選地，所述人CD8α鉸鏈區(qū)的氨基酸序列為SEQ ID NO.9，核酸序列為SEQ ID NO.10。

優(yōu)選地，所述跨膜區(qū)為人CD28跨膜區(qū)。

優(yōu)選地，所述人CD28跨膜區(qū)的氨基酸序列為SEQ ID NO.11，核酸序列為SEQ ID NO.12。

作為優(yōu)選技術(shù)方案，所述胞內(nèi)區(qū)為人CD3ζ胞內(nèi)區(qū)、人CD28胞內(nèi)區(qū)或人4-1BB胞內(nèi)區(qū)中的任意一種或至少兩種的組合，例如可以是CD3ζ胞內(nèi)區(qū)、人CD28胞內(nèi)區(qū)、人4-1BB胞內(nèi)區(qū)、人CD28胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)、人4-1BB胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)、人CD3ζ胞內(nèi)區(qū)與人4-1BB胞內(nèi)區(qū)的串聯(lián)或人CD28胞內(nèi)區(qū)、人4-1BB胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)，優(yōu)選為CD3ζ胞內(nèi)區(qū)、人CD28胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)、人4-1BB胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)或人CD28胞內(nèi)區(qū)、人4-1BB胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)。

優(yōu)選地，所述人CD3ζ胞內(nèi)區(qū)的氨基酸序列為SEQ ID NO.13，核酸序列為SEQ ID NO.14。

優(yōu)選地，所述人CD28胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)的氨基酸序列為SEQ ID NO.15，核酸序列為SEQ ID NO.16。

優(yōu)選地，所述人4-1BB胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)的氨基酸序列為SEQ ID NO.17，核酸序列為SEQ ID NO.18。

優(yōu)選地，所述人CD28胞內(nèi)區(qū)、人4-1BB胞內(nèi)區(qū)與人CD3ζ胞內(nèi)區(qū)的串聯(lián)的氨基酸序列為SEQ ID NO.19，核酸序列為SEQ ID NO.20。

優(yōu)選地，所述嵌合抗原受體的氨基末端含有一個人CD8α信號肽。

優(yōu)選地，所述人CD8α信號肽的氨基酸序列為SEQ ID NO.21，核酸序列為SEQ ID NO.22。

作為優(yōu)選技術(shù)方案，所述嵌合抗原受體由人CD8α信號肽、VAR2CSA、人CD8α鉸鏈區(qū)、人CD28跨膜區(qū)和胞內(nèi)區(qū)、人4-1BB胞內(nèi)區(qū)以及人CD3ζ胞內(nèi)區(qū)串聯(lián)構(gòu)成。

優(yōu)選地，所述嵌合抗原受體的氨基酸序列為SEQ ID NO.1，核酸序列為SEQ ID NO.2。

優(yōu)選地，所述的嵌合抗原受體通過其編碼的核酸序列轉(zhuǎn)染到T細胞中表達。

優(yōu)選地，所述轉(zhuǎn)染的方式為病毒載體、真核表達質(zhì)?；騧RNA序列中的任意一種或至少兩種的組合。

優(yōu)選地，所述病毒載體為慢病毒載體、腺病毒載體或逆轉(zhuǎn)錄病毒載體中的任意一種或至少兩種的組合。

第二方面，本發(fā)明提供一種組合物，所述組合物包括第一方面所述的嵌合抗原受體。

第三方面，本發(fā)明提供如第一方面所述的嵌合抗原受體和/或如第二方面所述的組合物在制備嵌合抗原受體T細胞及其在腫瘤治療藥物中的應用。

與現(xiàn)有技術(shù)相比，本發(fā)明具有如下有益效果：

(1)本發(fā)明的嵌合抗原受體，通過VAR2CSA識別pl-CS的方式，可以使CAR-T細胞特異的識別腫瘤細胞，更容易靶向到腫瘤微環(huán)境，進入到腫瘤組織內(nèi)部，可以更特異的識別腫瘤細胞，對腫瘤微環(huán)境具有更好的靶向性；

(2)本發(fā)明表達此嵌合抗原受體的T細胞能最大程度的殺傷腫瘤細胞，對正常組織的傷害程度很小，能突破腫瘤免疫抑制微環(huán)境，從而對實體瘤具有更好的療效，且?guī)缀跄苤委煾鞣N類型的腫瘤。

附圖說明

圖1為靶向pl-CS的CAR慢病毒表達載體示意圖；

圖2(A)為熒光顯微鏡下觀察慢病毒感染96h后T細胞的綠色熒光(GFP)表達情況，圖2(B)為流式細胞儀顯示慢病毒感染96h后T細胞的GFP陽性率；

圖3(A)為實驗組、陰性對照組、陽性對照組和野生型T細胞組上清中IL-2濃度比較，圖3(B)為實驗組、陰性對照組、陽性對照組和野生型T細胞組上清中IFN-γ濃度比較；

圖4為在不同效靶比例情況下，實驗組、陰性對照組和野生型T細胞組對B細胞淋巴瘤細胞系Raji的細胞毒性比較，其中，實驗組、陰性對照組合野生組的柱狀圖從左到右分別為效靶比8:1、4:1和1:1。

具體實施方式

為更進一步闡述本發(fā)明所采取的技術(shù)手段及其效果，以下結(jié)合附圖并通過具體實施方式來進一步說明本發(fā)明的技術(shù)方案，但本發(fā)明并非局限在實施例范圍內(nèi)。

實施例1：靶向pl-CS的CAR基因序列的確定

從GenBank數(shù)據(jù)庫以及文獻中搜索到瘧原蟲VAR2CSA基因、人CD8α信號肽基因、人CD8α鉸鏈區(qū)基因、人CD28跨膜區(qū)和胞內(nèi)區(qū)基因、人4-1BB胞內(nèi)區(qū)基因以及人CD3ζ胞內(nèi)區(qū)基因序列。將其進行密碼子優(yōu)化，保證在編碼氨基酸序列不變的情況下更適合人類細胞表達。

各基因序列信息見SEQUENCE LISTING(序列表SEQ ID NO.1-22)。

實施例2：慢病毒表達載體pLHlentiCAR004的構(gòu)建

將上述基因序列依次按人CD8α信號肽基因、VAR2CSA、人CD8α鉸鏈區(qū)基因、人CD28跨膜區(qū)和胞內(nèi)區(qū)基因、人4-1BB胞內(nèi)區(qū)基因以及人CD3ζ胞內(nèi)區(qū)基因序列進行連接，組成嵌合抗原受體，所述抗原受體的核酸序列如SEQ ID NO.2所示，引入合適的酶切位點，進行人工全基因合成，克隆到慢病毒表達載體中得到pLHlentiCAR004質(zhì)粒。質(zhì)粒示意圖見圖1。

將重組質(zhì)粒送上海英駿生物技術(shù)有限公司進行測序，測序結(jié)果與擬合成的序列比對以證實序列完全正確。

實施例3：慢病毒表達載體和包裝載體的大量提取

將pLHlentiCAR004質(zhì)粒以及psPAX2和pMD2.G包裝質(zhì)粒的stbl3大腸桿菌在LB培養(yǎng)液中大量培養(yǎng)，以堿裂解法大量提取質(zhì)粒，以備轉(zhuǎn)染，具體過程為：

1)分別將帶有pLHlentiCAR004、psPAX2、pMD2.G的stbl3大腸桿菌種接種于含有500mL LB/抗生素培養(yǎng)液的培養(yǎng)瓶中，37℃搖床培養(yǎng)12-16小時；

2)3000*g離心10min，收集菌體，倒掉培養(yǎng)基并反扣于吸水紙輕輕拍打以吸盡殘液。收集的菌體采用廣州美基生物科技有限公司MaxPure Plasmid Maxi Kit大提質(zhì)粒，提取質(zhì)粒的具體步驟為：

(1)加入20mL BufferP1/RNase A至菌體中，渦旋重懸細菌(確保細菌完全重懸，重懸后的溶液沒有細胞團塊)；向重懸液中加入20mL Buffer P2，顛倒混勻10-15次，靜置1-2min(一定要輕輕混勻，注意操作時間不能超過4分鐘)；

(2)向裂解液中加入20mL Buffer E3，立即顛倒混勻15-20次，混勻過程要輕柔而充分，中和充分的標準是：溶液變得不粘稠，沉淀物分散顯白色；3000*g離心10min，然后轉(zhuǎn)移上清液至新的離心管中，加入1/3倍體積Buffer E4至上清液中，渦旋混勻；

(3)將MaxPure Micro Column套在50mL離心管中，轉(zhuǎn)移15mL混合液至柱子中，3000*g離心10min；倒掉流出液，把柱子套回收集管中，繼續(xù)轉(zhuǎn)移剩下的溶液至柱子中，3000*g離心10分鐘；倒掉流出液，把柱子套回收集管中，加入20mL Buffer E5至柱子，3000*g離心10分鐘；倒掉濾液把柱子套回收集管中；

(4)加入20mL Buffer PW2(已用無水乙醇稀釋)至柱子，3000*g離心10分鐘；倒掉濾液把珠子套回收集管中，加入20mL Buffer PW2(已用無水乙醇稀釋)至柱子，3000*g離心10分鐘；倒棄濾液把柱子套回收集管中，4000*g離心10分鐘；

(5)把柱子套在滅菌的50mL離心管中，加入30-200mL滅菌水至柱子的膜中央，靜置2分鐘，10000*g離心5min，收集分離液；

(6)進一步抽提去除內(nèi)毒素，用滅菌水調(diào)整質(zhì)粒濃度在0.1g-0.6mg/mL之間；

(7)加入0.1倍體積Buffer ER1和0.1倍體積Buffer ER2至質(zhì)粒溶液中，顛倒混勻；冰上放置20min，期間顛倒混勻多次；42℃水浴5min后室溫在10000*g速度下離心3min；

(8)輕輕取出樣品，發(fā)現(xiàn)底部形成了一層紅色的溶液，小心把上清液轉(zhuǎn)移到離心管仲，加入0.7倍體積的異丙醇至上清液中，顛倒混勻多次；室溫下，在10000*g速度下離心10min，倒掉上清液，加入1倍體積的70％乙醇至沉淀中。渦旋混勻15-30s；室溫下，在10000*g速度下離心3min，小心倒掉上清液；室溫下，在10000*g速度下離心1min收集管壁上的液滴，不要吸到沉淀，在空氣中干燥10min；

(9)加入適量無菌水到質(zhì)粒中，渦旋10-20s混勻，室溫靜置30min讓質(zhì)粒充分溶解，期間顛倒混勻多次；

在Nanodrop紫外分光光度計上測量質(zhì)粒濃度，然后將質(zhì)粒保存在-20℃中。

實施例4：慢病毒的包裝、濃縮和滴定

1)慢病毒的包裝

(1)細胞預板：在轉(zhuǎn)染前24h，取長滿的293T細胞進行預板，以第二天能達到70％匯合度為宜(75mm培養(yǎng)皿內(nèi)預板500萬)。

(2)第二天：轉(zhuǎn)染

(a)觀察預板細胞的生長情況，以細胞的匯合度在90％左右為宜，吸去培養(yǎng)基，加入新鮮預熱的完全培養(yǎng)基(75mm培養(yǎng)皿內(nèi)15mL)；

(b)轉(zhuǎn)染前將轉(zhuǎn)染所需的PEI、質(zhì)粒、Opti-MEM I Reduced Serum Medium等放置室溫平衡；

(c)制備質(zhì)粒的混合液：本次包裝共10皿293T細胞，每一皿細胞準備混合液500μL，故需準備混合液總體積為5mL；先根據(jù)各種質(zhì)粒用量加入三種質(zhì)粒，共420μg(pLHlentiCAR004:psPAX2:pMD2.G＝3:2:1)，加入Opti-MEM I Reduced Serum Medium調(diào)整體積到2.5mL，吹打混勻后室溫放置10min；再取PEI和Opti-MEM I Reduced Serum Medium各1.25mL，混勻后室溫放置10min，最后將以上兩種溶液混合，混勻后室溫放置10min，即為轉(zhuǎn)染用的混合液；

(d)混合液逐滴加入培養(yǎng)皿中，每皿500μL；

(3)轉(zhuǎn)染6h以后，需把每皿培養(yǎng)基換成17mL UltraCLUTURE無血清培養(yǎng)基；

(4)第三至五天，病毒的收集：轉(zhuǎn)染48h-72h，分別收集培養(yǎng)上清，收集的病毒上清暫存于4℃。

2)慢病毒濃縮

將收集的培養(yǎng)上清用0.45um的膜過濾后用SW-32Ti的轉(zhuǎn)子25000rpm(對應離心力為106750g)，4℃離心2h，去上清后，用200uL的PBS重懸，保存于-80℃。

3)慢病毒的滴定

(1)6孔板中預鋪板293T細胞，3×10⁵cells/well，37℃5％CO₂培養(yǎng)箱中培養(yǎng)過夜(大約18-20h)；

(2)配制polybrene母液：滅菌純水，8mg/mL，0.22μm濾頭過濾，分裝，保存于-20℃?zhèn)溆茫?/p>

(3)配制含polybrene的DMEM培養(yǎng)基：10％FBS，1％雙抗，8μg/mL polybrene(培養(yǎng)基體積的千分之一)；

(4)融化病毒：從-80℃取出病毒凍存液，在室溫融化，融化后置于冰上拿進細胞間備用；

(5)稀釋病毒：用配好的含polybrene的5％FBS DMEM完全培養(yǎng)基將病毒原液進行10倍稀釋，得到10^-1-10^-4的病毒稀釋液，注意每次吸取病毒液進行下一步稀釋前要混勻；

(6)棄掉舊培養(yǎng)基，第一孔中加入1mL不含病毒的培養(yǎng)基作為陰性對照。其余每孔中加入1mL對應的病毒稀釋液，37℃培養(yǎng)18-20h；

(7)第二天早上去掉含病毒的培養(yǎng)基，替換以2mL不含聚凝胺的5％FBS DMEM培養(yǎng)基；顯微鏡下觀察細胞有表達GFP；

(8)吸去培養(yǎng)基，然后用胰酶消化細胞；將細胞吹成單個細胞，加入適量完全培養(yǎng)基終止胰酶反應，300g離心3min收集細胞；

(9)倒去上清，用適量預冷PBS重懸后，300g離心3min，收集細胞，再重復一次；

(10)最后用1mL預冷PBS重懸，置于冰上，用于流式分析。

(11)選擇GFP陽性率在1％-20％的孔，各孔分別計算病毒滴度，最后取平均值；

計算公式：titer＝{(F×Cn)/V}×DF，單位為TU/mL；

其中，F(xiàn)：GFP陽性細胞率；Cn：每孔鋪板的細胞數(shù)量；V：所加入病毒稀釋液體積；DF：病毒稀釋倍數(shù)

計算得到病毒滴度為：1.6×10⁸-1×10¹⁰之間。

實施例5：T細胞的分離，培養(yǎng)和感染

1)PBMC細胞的分離

(1)采血50mL，800g離心10min(加速設置為3，降速設置為4)；

(2)取白細胞層，用2％FBS稀釋至8mL；

(3)吸取4mL LymphoPrep加入15mL離心管中，再將稀釋血液小心沿管壁加至分層液上，保持兩者界面清晰；

(4)800g離心20min(加速設置為6，降速設置為1)；

(5)用巴氏吸管輕輕吸出灰白色的單個核細胞，加入另一支已含有10mL RF-10的離心管中，混勻；

(6)以500g離心5min，棄上清；

(7)加10mL RF-10重懸細胞，trypan blue計數(shù)，350g離心10min，棄上清。

2)磁珠分選CD4+T(CD8+T)細胞

(1)渦旋混勻磁珠，取25μL磁珠于試管中，加入1mL Buffer 1混勻，將試管放在磁力架上1min，去上清。加25μL Bffer1重懸磁珠備用；

(2)PBMC用Buffer 1重懸至密度為107個/mL；

(3)向1mL的PBMC中加入25μL洗滌的磁珠，2-8℃孵育20min，放在搖床上傾斜旋轉(zhuǎn)；

(4)將試管放在磁鐵上2min，收集上清；

(5)移開試管，加1mL Buffer 1，吹打混勻，放在磁珠上2min，收集上清，重復一次；

(6)加100μL Buffer 2重懸細胞，加10μL DETCHaBEAD，室溫下孵育45min使細胞從磁珠上釋放；

(7)將試管放在磁力架上1min，含有細胞的上清轉(zhuǎn)移到新的試管中，加500μL Buffer 2洗滌磁珠2-3次，收集上清；

(8)加4mL Buffer 2，350g離心5min，去上清，用Buffer 2重懸細胞；

(9)分選CD4+T細胞過程中收集的上清按試劑盒操作收集CD8+T細胞。

3)T細胞的培養(yǎng)

(1)磁珠分選后的CD4+T(CD8+T)細胞350g離心10min；

(2)RF-10重懸計數(shù)；

(3)按照1：1的比例加CD4+T和CD8+T細胞培養(yǎng)板中培養(yǎng)，細胞密度為5×10⁵個/mL；

(4)在T細胞專用培養(yǎng)基加入CD3/CD28抗體磁珠，加入磁珠的量按與細胞的比例1:1加入；

(5)加rhIL-2，使終濃度為10ng/mL；

(6)每周計數(shù)2-3次，記錄細胞的增殖情況。

4)T細胞的慢病毒轉(zhuǎn)染。

(1)磁珠分選后的CD4+T(CD8+T)細胞350g離心10min，加完全培養(yǎng)基重懸計數(shù)；

(2)按照1：1的比例加CD4+T和CD8+T細胞于96孔板中培養(yǎng)，細胞密度為5×10⁵個/mL，每孔細胞數(shù)為1×10⁵個；

(3)洗滌磁珠，向培養(yǎng)皿中加入與細胞1:1比例的磁珠；

(4)加rhIL-2，使終濃度為10μg/L；

(5)刺激24h后，感染細胞，加polybrene，使終濃度為6μg/mL，混勻；

(6)16-24h后換液；

(7)3天后進行熒光顯微鏡拍照。

熒光顯微鏡的結(jié)果圖如圖2(A)，流式檢測感染效率如圖2(B)所示，可見，流式檢測的感染效率為50％左右，可以高效制得CAR-T細胞。

實施例6：CAR-T細胞體外細胞因子釋放及檢測

(1)48孔板中，每孔CAR-T細胞1.5×10⁵個與Huh7細胞每孔1.5×10⁵個共培養(yǎng)24h，加500μL RPMI-1640培養(yǎng)基(不含血清和rhIL-2)；

(2)樣品收集和儲存：離心收集上清，直接開始實驗或分裝保存于-20℃(100μL每管)；

(3)試劑準備：使用前將所有試劑拿出恢復至室溫，用去離子水將Wash Buffer濃縮液稀釋至500mL；底物顯色液，將A與B等體積混合，15min內(nèi)用完，每孔200μL混合液；細胞因子標準品，去500μL儲存液，梯度濃度稀釋6組；

(4)將樣品以及濃度梯度稀釋的標準品加入已包被抗體的ELISA孔板中，100μL每孔，除去氣泡室溫孵育2h(樣品每組設三個復孔)；

(5)移去孔內(nèi)液體，每孔加300μLWB洗滌，洗滌三次，最后一次吸干WB；

(6)加200μL對應的檢測抗體，室溫孵育2h；

(7)移去孔內(nèi)液體，每孔加300μLWB洗滌，洗滌三次，最后一次吸干WB；

(8)加200μL底物液，室溫孵育20min(避光)；

(9)加50μL終止液，溶液由藍色變?yōu)辄S色，如果顏色為綠色或者沒有改變顏色，輕拍使其混勻；

(10)30min內(nèi)檢測450nm處的吸光度；

(11)根據(jù)標準曲線計數(shù)各細胞因子濃度，

結(jié)果如圖3(A)和3(B)所示，可見，CAR-T細胞與腫瘤細胞共培養(yǎng)不會釋放細胞因子，表明CAR-T細胞不會具有強烈的細胞因子風暴有害作用，也就不會引起炎癥反應的副作用。

實施例7：CAR-T細胞體外殺傷腫瘤細胞

1)確定最佳的細胞鋪板濃度

(1)收集各腫瘤細胞，用分析培養(yǎng)基洗滌，調(diào)整細胞濃度為1×10⁵個/mL；

(2)在96孔板中加100μL分析用培養(yǎng)基，鋪滿；

(3)加100μL細胞懸液于96孔板中，依次稀釋，最后一個濃度梯度吸出100μL細胞懸液。設置六個復孔，三孔為High control組，三孔為Low control組，并設置三孔無細胞的分析培養(yǎng)基作為Background control(量程，500-50000)；

(4)培養(yǎng)箱孵育18h；

(5)HC組每孔加入5μL細胞溶解液，孵育15min，震動加速細胞溶解；

(6)每孔加入100μL現(xiàn)配的反應液，15℃-25℃孵育30min(注意96孔板避光)；

(7)每孔加入50μL終止液，震動10s；

(8)490nm測吸光度；

(9)分析數(shù)據(jù)，確定最佳細胞濃度(HC與LC的最大差值)。

2)細胞介導的毒性檢測

(1)收集CAR-T細胞，用分析培養(yǎng)基洗滌細胞；

(2)根據(jù)不同的效靶比和1)中結(jié)果的最佳濃度來確定鋪于96孔板中的CAR-T細胞數(shù)；

(3)在96孔板中鋪的相應細胞懸液每孔體積調(diào)為50μL；

(4)收集各腫瘤細胞，調(diào)整細胞濃度為最佳濃度的2倍，加50μL細胞懸液于T細胞中；

(5)設置不同對照組；

(6)37度培養(yǎng)箱孵育18h；

(7)每孔加入5μL細胞溶解液，孵育15min，震動加速細胞溶解；

(8)每孔加入100μL現(xiàn)配的反應液，15℃-25℃孵育30min(注意96孔板避光)；

(9)每孔加入50μL終止液，震動10s；

(10)490nm測吸光度；

(11)計算每個樣品細胞毒性的百分比。

結(jié)果如圖4所示，不同效靶比的CAR-T細胞具有對腫瘤細胞的毒性，能高效的殺傷腫瘤細胞，釋放LDH，作為對照組的野生型T細胞，不能殺傷腫瘤細胞。

綜上所述，本發(fā)明表達此嵌合抗原受體的T細胞能最大程度的殺傷腫瘤細胞，不釋放細胞因子，對正常組織的傷害很小，能突破腫瘤免疫抑制微環(huán)境，從而對實體瘤具有更好的療效，且?guī)缀跄苤委煾鞣N類型的腫瘤。

申請人聲明，本發(fā)明通過上述實施例來說明本發(fā)明的詳細方法，但本發(fā)明并不局限于上述詳細方法，即不意味著本發(fā)明必須依賴上述詳細方法才能實施。所屬技術(shù)領(lǐng)域的技術(shù)人員應該明了，對本發(fā)明的任何改進，對本發(fā)明產(chǎn)品各原料的等效替換及輔助成分的添加、具體方式的選擇等，均落在本發(fā)明的保護范圍和公開范圍之內(nèi)。

序列表

<110> 廣州中科藍華生物科技有限公司

<120> 一種抗胎盤樣硫酸軟骨素的嵌合抗原受體及其應用

<130> 2015

<160> 22

<170> PatentIn version 3.3

<210> 1

<211> 900

<212> PRT

<213> 人工合成序列

<400> 1

Asn Tyr Ile Lys Gly Asp Pro Tyr Phe Ala Glu Tyr Ala Thr Lys Leu

1 5 10 15

Ser Phe Ile Leu Asn Ser Ser Asp Ala Asn Asn Pro Ser Glu Lys Ile

20 25 30

Gln Lys Asn Asn Asp Glu Val Cys Asn Cys Asn Glu Ser Gly Ile Ala

35 40 45

Ser Val Glu Gln Glu Gln Ile Ser Asp Pro Ser Ser Asn Lys Thr Cys

50 55 60

Ile Thr His Ser Ser Ile Lys Ala Asn Lys Lys Lys Val Cys Lys His

65 70 75 80

Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Arg Val Cys Val

85 90 95

Ile Glu His Thr Ser Leu Ser Gly Val Glu Asn Cys Cys Cys Gln Asp

100 105 110

Phe Leu Arg Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Ser Gly Ser

115 120 125

Ser Ser Asn Gly Ser Cys Asn Asn Lys Asn Gln Glu Ala Cys Glu Lys

130 135 140

Asn Leu Glu Lys Val Leu Ala Ser Leu Thr Asn Cys Tyr Lys Cys Asp

145 150 155 160

Lys Cys Lys Ser Glu Gln Ser Lys Lys Asn Asn Lys Asn Trp Ile Trp

165 170 175

Lys Lys Ser Ser Gly Lys Glu Gly Gly Leu Gln Lys Glu Tyr Ala Asn

180 185 190

Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Cys Leu Val Val Cys

195 200 205

Leu Asp Glu Lys Gly Lys Lys Thr Gln Glu Leu Lys Asn Ile Arg Thr

210 215 220

Asn Ser Glu Leu Leu Lys Glu Trp Ile Ile Ala Ala Phe His Glu Gly

225 230 235 240

Lys Asn Leu Lys Pro Ser His Glu Lys Lys Asn Asp Asp Asn Gly Lys

245 250 255

Lys Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr Gly Asp Leu

260 265 270

Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys Asp Leu Glu

275 280 285

Leu Asn Leu Gln Lys Ile Phe Gly Lys Leu Phe Arg Lys Tyr Ile Lys

290 295 300

Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser Leu Asp Glu

305 310 315 320

Leu Arg Glu Ser Trp Trp Asn Thr Asn Lys Lys Tyr Ile Trp Leu Ala

325 330 335

Met Lys His Gly Ala Gly Met Asn Ser Thr Thr Cys Cys Gly Asp Gly

340 345 350

Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro Thr Ile Asp

355 360 365

Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val Glu His Phe

370 375 380

Cys Lys Gln Arg Gln Glu Lys Val Lys Pro Val Ile Glu Asn Cys Lys

385 390 395 400

Ser Cys Lys Glu Ser Gly Gly Thr Cys Asn Gly Glu Cys Lys Thr Glu

405 410 415

Cys Lys Asn Lys Cys Glu Val Tyr Lys Lys Phe Ile Glu Asp Cys Lys

420 425 430

Gly Gly Asp Gly Thr Ala Gly Ser Ser Trp Val Lys Arg Trp Asp Gln

435 440 445

Ile Tyr Lys Arg Tyr Ser Lys Tyr Ile Glu Asp Ala Lys Arg Asn Arg

450 455 460

Lys Ala Gly Thr Lys Asn Cys Gly Pro Ser Ser Thr Thr Asn Ala Ala

465 470 475 480

Glu Asn Lys Cys Val Gln Ser Asp Ile Asp Ser Phe Phe Lys His Leu

485 490 495

Ile Asp Ile Gly Leu Thr Thr Pro Ser Ser Tyr Leu Ser Ile Val Leu

500 505 510

Asp Asp Asn Ile Cys Gly Ala Asp Lys Ala Pro Trp Thr Thr Tyr Thr

515 520 525

Thr Tyr Thr Thr Thr Glu Lys Cys Asn Lys Glu Thr Asp Lys Ser Lys

530 535 540

Leu Gln Gln Cys Asn Thr Ala Val Val Val Asn Val Pro Ser Pro Leu

545 550 555 560

Gly Asn Thr Pro His Gly Tyr Lys Tyr Ala Cys Gln Cys Lys Ile Pro

565 570 575

Thr Asn Glu Glu Thr Cys Asp Asp Arg Lys Glu Tyr Met Asn Gln Trp

580 585 590

Ser Cys Gly Ser Ala Arg Thr Met Lys Arg Gly Tyr Lys Asn Asp Asn

595 600 605

Tyr Glu Leu Cys Lys Tyr Asn Gly Val Asp Val Lys Pro Thr Thr Val

610 615 620

Arg Ser Asn Ser Ser Lys Leu Asp Thr Thr Thr Pro Ala Pro Arg Pro

625 630 635 640

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

645 650 655

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

660 665 670

Asp Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu

675 680 685

Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

690 695 700

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

705 710 715 720

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

725 730 735

Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu

740 745 750

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

755 760 765

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

770 775 780

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

785 790 795 800

Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg

805 810 815

Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met

820 825 830

Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn

835 840 845

Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met

850 855 860

Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly

865 870 875 880

Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala

885 890 895

Leu Pro Pro Arg

900

<210> 2

<211> 2700

<212> DNA

<213> 人工合成序列

<400> 2

aactacatca agggcgaccc ctacttcgcc gagtacgcca ccaagctgag cttcatcctg 60

aacagcagcg acgccaacaa ccccagcgag aagatccaga agaacaacga cgaggtgtgc 120

aactgcaacg agagcggcat cgccagcgtg gagcaggagc agatcagcga ccccagcagc 180

aacaagacct gcatcaccca cagcagcatc aaggccaaca agaagaaggt gtgcaagcac 240

gtgaagctgg gcgtgcgcga gaacgacaag gacctgcgcg tgtgcgtgat cgagcacacc 300

agcctgagcg gcgtggagaa ctgctgctgc caggacttcc tgcgcatcct gcaggagaac 360

tgcagcgaca acaagagcgg cagcagcagc aacggcagct gcaacaacaa gaaccaggag 420

gcctgcgaga agaacctgga gaaggtgctg gccagcctga ccaactgcta caagtgcgac 480

aagtgcaaga gcgagcagag caagaagaac aacaagaact ggatctggaa gaagagcagc 540

ggcaaggagg gcggcctgca gaaggagtac gccaacacca tcggcctgcc cccccgcacc 600

cagagcctgt gcctggtggt gtgcctggac gagaagggca agaagaccca ggagctgaag 660

aacatccgca ccaacagcga gctgctgaag gagtggatca tcgccgcctt ccacgagggc 720

aagaacctga agcccagcca cgagaagaag aacgacgaca acggcaagaa gctgtgcaag 780

gccctggagt acagcttcgc cgactacggc gacctgatca agggcaccag catctgggac 840

aacgagtaca ccaaggacct ggagctgaac ctgcagaaga tcttcggcaa gctgttccgc 900

aagtacatca agaagaacaa caccgccgag caggacacca gctacagcag cctggacgag 960

ctgcgcgaga gctggtggaa caccaacaag aagtacatct ggctggccat gaagcacggc 1020

gccggcatga acagcaccac ctgctgcggc gacggcagcg tgaccggcag cggcagcagc 1080

tgcgacgaca tccccaccat cgacctgatc ccccagtacc tgcgcttcct gcaggagtgg 1140

gtggagcact tctgcaagca gcgccaggag aaggtgaagc ccgtgatcga gaactgcaag 1200

agctgcaagg agagcggcgg cacctgcaac ggcgagtgca agaccgagtg caagaacaag 1260

tgcgaggtgt acaagaagtt catcgaggac tgcaagggcg gcgacggcac cgccggcagc 1320

agctgggtga agcgctggga ccagatctac aagcgctaca gcaagtacat cgaggacgcc 1380

aagcgcaacc gcaaggccgg caccaagaac tgcggcccca gcagcaccac caacgccgcc 1440

gagaacaagt gcgtgcagag cgacatcgac agcttcttca agcacctgat cgacatcggc 1500

ctgaccaccc ccagcagcta cctgagcatc gtgctggacg acaacatctg cggcgccgac 1560

aaggccccct ggaccaccta caccacctac accaccaccg agaagtgcaa caaggagacc 1620

gacaagagca agctgcagca gtgcaacacc gccgtggtgg tgaacgtgcc cagccccctg 1680

ggcaacaccc cccacggcta caagtacgcc tgccagtgca agatccccac caacgaggag 1740

acctgcgacg accgcaagga gtacatgaac cagtggagct gcggcagcgc ccgcaccatg 1800

aagcgcggct acaagaacga caactacgag ctgtgcaagt acaacggcgt ggacgtgaag 1860

cccaccaccg tgcgcagcaa cagcagcaag ctggacacca ccacccccgc cccccgcccc 1920

cccacccccg cccccaccat cgccagccag cccctgagcc tgcgccccga ggcctgccgc 1980

cccgccgccg gcggcgccgt gcacacccgc ggcctggact tcgcctgcga cttctgggtg 2040

ctggtggtgg tgggcggcgt gctggcctgc tacagcctgc tggtgaccgt ggccttcatc 2100

atcttctggg tgcgcagcaa gcgcagccgc ctgctgcaca gcgactacat gaacatgacc 2160

ccccgccgcc ccggccccac ccgcaagcac taccagccct acgccccccc ccgcgacttc 2220

gccgcctacc gcagcaagcg cggccgcaag aagctgctgt acatcttcaa gcagcccttc 2280

atgcgccccg tgcagaccac ccaggaggag gacggctgca gctgccgctt ccccgaggag 2340

gaggagggcg gctgcgagct gcgcgtgaag ttcagccgca gcgccgacgc ccccgcctac 2400

cagcagggcc agaaccagct gtacaacgag ctgaacctgg gccgccgcga ggagtacgac 2460

gtgctggaca agcgccgcgg ccgcgacccc gagatgggcg gcaagcccca gcgccgcaag 2520

aacccccagg agggcctgta caacgagctg cagaaggaca agatggccga ggcctacagc 2580

gagatcggca tgaagggcga gcgccgccgc ggcaagggcc acgacggcct gtaccagggc 2640

ctgagcaccg ccaccaagga cacctacgac gccctgcaca tgcaggccct gcccccccgc 2700

<210> 3

<211> 2646

<212> PRT

<213> 人工合成序列

<400> 3

Met Asp Lys Ser Ser Ile Ala Asn Lys Ile Glu Ala Tyr Leu Gly Ala

1 5 10 15

Lys Ser Asp Asp Ser Lys Ile Asp Gln Ser Leu Lys Ala Asp Pro Ser

20 25 30

Glu Val Gln Tyr Tyr Gly Ser Gly Gly Asp Gly Tyr Tyr Leu Arg Lys

35 40 45

Asn Ile Cys Lys Ile Thr Val Asn His Ser Asp Ser Gly Thr Asn Asp

50 55 60

Pro Cys Asp Arg Ile Pro Pro Pro Tyr Gly Asp Asn Asp Gln Trp Lys

65 70 75 80

Cys Ala Ile Ile Leu Ser Lys Val Ser Glu Lys Pro Glu Asn Val Phe

85 90 95

Val Pro Pro Arg Arg Gln Arg Met Cys Ile Asn Asn Leu Glu Lys Leu

100 105 110

Asn Val Asp Lys Ile Arg Asp Lys His Ala Phe Leu Ala Asp Val Leu

115 120 125

Leu Thr Ala Arg Asn Glu Gly Glu Arg Ile Val Gln Asn His Pro Asp

130 135 140

Thr Asn Ser Ser Asn Val Cys Asn Ala Leu Glu Arg Ser Phe Ala Asp

145 150 155 160

Ile Ala Asp Ile Ile Arg Gly Thr Asp Leu Trp Lys Gly Thr Asn Ser

165 170 175

Asn Leu Glu Gln Asn Leu Lys Gln Met Phe Ala Lys Ile Arg Glu Asn

180 185 190

Asp Lys Val Leu Gln Asp Lys Tyr Pro Lys Asp Gln Asn Tyr Arg Lys

195 200 205

Leu Arg Glu Asp Trp Trp Asn Ala Asn Arg Gln Lys Val Trp Glu Val

210 215 220

Ile Thr Cys Gly Ala Arg Ser Asn Asp Leu Leu Ile Lys Arg Gly Trp

225 230 235 240

Arg Thr Ser Gly Lys Ser Asn Gly Asp Asn Lys Leu Glu Leu Cys Arg

245 250 255

Lys Cys Gly His Tyr Glu Glu Lys Val Pro Thr Lys Leu Asp Tyr Val

260 265 270

Pro Gln Phe Leu Arg Trp Leu Thr Glu Trp Ile Glu Asp Phe Tyr Arg

275 280 285

Glu Lys Gln Asn Leu Ile Asp Asp Met Glu Arg His Arg Glu Glu Cys

290 295 300

Thr Ser Glu Asp His Lys Ser Lys Glu Gly Thr Ser Tyr Cys Ser Thr

305 310 315 320

Cys Lys Asp Lys Cys Lys Lys Tyr Cys Glu Cys Val Lys Lys Trp Lys

325 330 335

Ser Glu Trp Glu Asn Gln Lys Asn Lys Tyr Thr Glu Leu Tyr Gln Gln

340 345 350

Asn Lys Asn Glu Thr Ser Gln Lys Asn Thr Ser Arg Tyr Asp Asp Tyr

355 360 365

Val Lys Asp Phe Phe Lys Lys Leu Glu Ala Asn Tyr Ser Ser Leu Glu

370 375 380

Asn Tyr Ile Lys Gly Asp Pro Tyr Phe Ala Glu Tyr Ala Thr Lys Leu

385 390 395 400

Ser Phe Ile Leu Asn Ser Ser Asp Ala Asn Asn Pro Ser Glu Lys Ile

405 410 415

Gln Lys Asn Asn Asp Glu Val Cys Asn Cys Asn Glu Ser Gly Ile Ala

420 425 430

Ser Val Glu Gln Glu Gln Ile Ser Asp Pro Ser Ser Asn Lys Thr Cys

435 440 445

Ile Thr His Ser Ser Ile Lys Ala Asn Lys Lys Lys Val Cys Lys His

450 455 460

Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Arg Val Cys Val

465 470 475 480

Ile Glu His Thr Ser Leu Ser Gly Val Glu Asn Cys Cys Cys Gln Asp

485 490 495

Phe Leu Arg Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Ser Gly Ser

500 505 510

Ser Ser Asn Gly Ser Cys Asn Asn Lys Asn Gln Glu Ala Cys Glu Lys

515 520 525

Asn Leu Glu Lys Val Leu Ala Ser Leu Thr Asn Cys Tyr Lys Cys Asp

530 535 540

Lys Cys Lys Ser Glu Gln Ser Lys Lys Asn Asn Lys Asn Trp Ile Trp

545 550 555 560

Lys Lys Ser Ser Gly Lys Glu Gly Gly Leu Gln Lys Glu Tyr Ala Asn

565 570 575

Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Cys Leu Val Val Cys

580 585 590

Leu Asp Glu Lys Gly Lys Lys Thr Gln Glu Leu Lys Asn Ile Arg Thr

595 600 605

Asn Ser Glu Leu Leu Lys Glu Trp Ile Ile Ala Ala Phe His Glu Gly

610 615 620

Lys Asn Leu Lys Pro Ser His Glu Lys Lys Asn Asp Asp Asn Gly Lys

625 630 635 640

Lys Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr Gly Asp Leu

645 650 655

Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys Asp Leu Glu

660 665 670

Leu Asn Leu Gln Lys Ile Phe Gly Lys Leu Phe Arg Lys Tyr Ile Lys

675 680 685

Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser Leu Asp Glu

690 695 700

Leu Arg Glu Ser Trp Trp Asn Thr Asn Lys Lys Tyr Ile Trp Leu Ala

705 710 715 720

Met Lys His Gly Ala Gly Met Asn Ser Thr Thr Cys Cys Gly Asp Gly

725 730 735

Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro Thr Ile Asp

740 745 750

Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val Glu His Phe

755 760 765

Cys Lys Gln Arg Gln Glu Lys Val Lys Pro Val Ile Glu Asn Cys Lys

770 775 780

Ser Cys Lys Glu Ser Gly Gly Thr Cys Asn Gly Glu Cys Lys Thr Glu

785 790 795 800

Cys Lys Asn Lys Cys Glu Val Tyr Lys Lys Phe Ile Glu Asp Cys Lys

805 810 815

Gly Gly Asp Gly Thr Ala Gly Ser Ser Trp Val Lys Arg Trp Asp Gln

820 825 830

Ile Tyr Lys Arg Tyr Ser Lys Tyr Ile Glu Asp Ala Lys Arg Asn Arg

835 840 845

Lys Ala Gly Thr Lys Asn Cys Gly Pro Ser Ser Thr Thr Asn Ala Ala

850 855 860

Glu Asn Lys Cys Val Gln Ser Asp Ile Asp Ser Phe Phe Lys His Leu

865 870 875 880

Ile Asp Ile Gly Leu Thr Thr Pro Ser Ser Tyr Leu Ser Ile Val Leu

885 890 895

Asp Asp Asn Ile Cys Gly Ala Asp Lys Ala Pro Trp Thr Thr Tyr Thr

900 905 910

Thr Tyr Thr Thr Thr Glu Lys Cys Asn Lys Glu Thr Asp Lys Ser Lys

915 920 925

Leu Gln Gln Cys Asn Thr Ala Val Val Val Asn Val Pro Ser Pro Leu

930 935 940

Gly Asn Thr Pro His Gly Tyr Lys Tyr Ala Cys Gln Cys Lys Ile Pro

945 950 955 960

Thr Asn Glu Glu Thr Cys Asp Asp Arg Lys Glu Tyr Met Asn Gln Trp

965 970 975

Ser Cys Gly Ser Ala Arg Thr Met Lys Arg Gly Tyr Lys Asn Asp Asn

980 985 990

Tyr Glu Leu Cys Lys Tyr Asn Gly Val Asp Val Lys Pro Thr Thr Val

995 1000 1005

Arg Ser Asn Ser Ser Lys Leu Asp Asp Lys Asp Val Thr Phe Phe

1010 1015 1020

Asn Leu Phe Glu Gln Trp Asn Lys Glu Ile Gln Tyr Gln Ile Glu

1025 1030 1035

Gln Tyr Met Thr Asn Thr Lys Ile Ser Cys Asn Asn Glu Lys Asn

1040 1045 1050

Val Leu Ser Arg Val Ser Asp Glu Ala Ala Gln Pro Lys Phe Ser

1055 1060 1065

Asp Asn Glu Arg Asp Arg Asn Ser Ile Thr His Glu Asp Lys Asn

1070 1075 1080

Cys Lys Glu Lys Cys Lys Cys Tyr Ser Leu Trp Ile Glu Lys Ile

1085 1090 1095

Asn Asp Gln Trp Asp Lys Gln Lys Asp Asn Tyr Asn Lys Phe Gln

1100 1105 1110

Arg Lys Gln Ile Tyr Asp Ala Asn Lys Gly Ser Gln Asn Lys Lys

1115 1120 1125

Val Val Ser Leu Ser Asn Phe Leu Phe Phe Ser Cys Trp Glu Glu

1130 1135 1140

Tyr Ile Gln Lys Tyr Phe Asn Gly Asp Trp Ser Lys Ile Lys Asn

1145 1150 1155

Ile Gly Ser Asp Thr Phe Glu Phe Leu Ile Lys Lys Cys Gly Asn

1160 1165 1170

Asp Ser Gly Asp Gly Glu Thr Ile Phe Ser Glu Lys Leu Asn Asn

1175 1180 1185

Ala Glu Lys Lys Cys Lys Glu Asn Glu Ser Thr Asn Asn Lys Met

1190 1195 1200

Lys Ser Ser Glu Thr Ser Cys Asp Cys Ser Glu Pro Ile Tyr Ile

1205 1210 1215

Arg Gly Cys Gln Pro Lys Ile Tyr Asp Gly Lys Ile Phe Pro Gly

1220 1225 1230

Lys Gly Gly Glu Lys Gln Trp Ile Cys Lys Asp Thr Ile Ile His

1235 1240 1245

Gly Asp Thr Asn Gly Ala Cys Ile Pro Pro Arg Thr Gln Asn Leu

1250 1255 1260

Cys Val Gly Glu Leu Trp Asp Lys Arg Tyr Gly Gly Arg Ser Asn

1265 1270 1275

Ile Lys Asn Asp Thr Lys Glu Ser Leu Lys Gln Lys Ile Lys Asn

1280 1285 1290

Ala Ile Gln Lys Glu Thr Glu Leu Leu Tyr Glu Tyr His Asp Lys

1295 1300 1305

Gly Thr Ala Ile Ile Ser Arg Asn Pro Met Lys Gly Gln Lys Glu

1310 1315 1320

Lys Glu Glu Lys Asn Asn Asp Ser Asn Gly Leu Pro Lys Gly Phe

1325 1330 1335

Cys His Ala Val Gln Arg Ser Phe Ile Asp Tyr Lys Asn Met Ile

1340 1345 1350

Leu Gly Thr Ser Val Asn Ile Tyr Glu Tyr Ile Gly Lys Leu Gln

1355 1360 1365

Glu Asp Ile Lys Lys Ile Ile Glu Lys Gly Thr Thr Lys Gln Asn

1370 1375 1380

Gly Lys Thr Val Gly Ser Gly Ala Glu Asn Val Asn Ala Trp Trp

1385 1390 1395

Lys Gly Ile Glu Gly Glu Met Trp Asp Ala Val Arg Cys Ala Ile

1400 1405 1410

Thr Lys Ile Asn Lys Lys Gln Lys Lys Asn Gly Thr Phe Ser Ile

1415 1420 1425

Asp Glu Cys Gly Ile Phe Pro Pro Thr Gly Asn Asp Glu Asp Gln

1430 1435 1440

Ser Val Ser Trp Phe Lys Glu Trp Ser Glu Gln Phe Cys Ile Glu

1445 1450 1455

Arg Leu Gln Tyr Glu Lys Asn Ile Arg Asp Ala Cys Thr Asn Asn

1460 1465 1470

Gly Gln Gly Asp Lys Ile Gln Gly Asp Cys Lys Arg Lys Cys Glu

1475 1480 1485

Glu Tyr Lys Lys Tyr Ile Ser Glu Lys Lys Gln Glu Trp Asp Lys

1490 1495 1500

Gln Lys Thr Lys Tyr Glu Asn Lys Tyr Val Gly Lys Ser Ala Ser

1505 1510 1515

Asp Leu Leu Lys Glu Asn Tyr Pro Glu Cys Ile Ser Ala Asn Phe

1520 1525 1530

Asp Phe Ile Phe Asn Asp Asn Ile Glu Tyr Lys Thr Tyr Tyr Pro

1535 1540 1545

Tyr Gly Asp Tyr Ser Ser Ile Cys Ser Cys Glu Gln Val Lys Tyr

1550 1555 1560

Tyr Glu Tyr Asn Asn Ala Glu Lys Lys Asn Asn Lys Ser Leu Cys

1565 1570 1575

His Glu Lys Gly Asn Asp Arg Thr Trp Ser Lys Lys Tyr Ile Lys

1580 1585 1590

Lys Leu Glu Asn Gly Arg Thr Leu Glu Gly Val Tyr Val Pro Pro

1595 1600 1605

Arg Arg Gln Gln Leu Cys Leu Tyr Glu Leu Phe Pro Ile Ile Ile

1610 1615 1620

Lys Asn Lys Asn Asp Ile Thr Asn Ala Lys Lys Glu Leu Leu Glu

1625 1630 1635

Thr Leu Gln Ile Val Ala Glu Arg Glu Ala Tyr Tyr Leu Trp Lys

1640 1645 1650

Gln Tyr His Ala His Asn Asp Thr Thr Tyr Leu Ala His Lys Lys

1655 1660 1665

Ala Cys Cys Ala Ile Arg Gly Ser Phe Tyr Asp Leu Glu Asp Ile

1670 1675 1680

Ile Lys Gly Asn Asp Leu Val His Asp Glu Tyr Thr Lys Tyr Ile

1685 1690 1695

Asp Ser Lys Leu Asn Glu Ile Phe Asp Ser Ser Asn Lys Asn Asp

1700 1705 1710

Ile Glu Thr Lys Arg Ala Arg Thr Asp Trp Trp Glu Asn Glu Ala

1715 1720 1725

Ile Ala Val Pro Asn Ile Thr Gly Ala Asn Lys Ser Asp Pro Lys

1730 1735 1740

Thr Ile Arg Gln Leu Val Trp Asp Ala Met Gln Ser Gly Val Arg

1745 1750 1755

Lys Ala Ile Asp Glu Glu Lys Glu Lys Lys Lys Pro Asn Glu Asn

1760 1765 1770

Phe Pro Pro Cys Met Gly Val Gln His Ile Gly Ile Ala Lys Pro

1775 1780 1785

Gln Phe Ile Arg Trp Leu Glu Glu Trp Thr Asn Glu Phe Cys Glu

1790 1795 1800

Lys Tyr Thr Lys Tyr Phe Glu Asp Met Lys Ser Asn Cys Asn Leu

1805 1810 1815

Arg Lys Gly Ala Asp Asp Cys Asp Asp Asn Ser Asn Ile Glu Cys

1820 1825 1830

Lys Lys Ala Cys Ala Asn Tyr Thr Asn Trp Leu Asn Pro Lys Arg

1835 1840 1845

Ile Glu Trp Asn Gly Met Ser Asn Tyr Tyr Asn Lys Ile Tyr Arg

1850 1855 1860

Lys Ser Asn Lys Glu Ser Glu Asp Gly Lys Asp Tyr Ser Met Ile

1865 1870 1875

Met Glu Pro Thr Val Ile Asp Tyr Leu Asn Lys Arg Cys Asn Gly

1880 1885 1890

Glu Ile Asn Gly Asn Tyr Ile Cys Cys Ser Cys Lys Asn Ile Gly

1895 1900 1905

Glu Asn Ser Thr Ser Gly Thr Val Asn Lys Lys Leu Gln Lys Lys

1910 1915 1920

Glu Thr Gln Cys Glu Asp Asn Lys Gly Pro Leu Asp Leu Met Asn

1925 1930 1935

Lys Val Leu Asn Lys Met Asp Pro Lys Tyr Ser Glu His Lys Met

1940 1945 1950

Lys Cys Thr Glu Val Tyr Leu Glu His Val Glu Glu Gln Leu Lys

1955 1960 1965

Glu Ile Asp Asn Ala Ile Lys Asp Tyr Lys Leu Tyr Pro Leu Asp

1970 1975 1980

Arg Cys Phe Asp Asp Lys Ser Lys Met Lys Val Cys Asp Leu Ile

1985 1990 1995

Gly Asp Ala Ile Gly Cys Lys His Lys Thr Lys Leu Asp Glu Leu

2000 2005 2010

Asp Glu Trp Asn Asp Val Asp Met Arg Asp Pro Tyr Asn Lys Tyr

2015 2020 2025

Lys Gly Val Leu Ile Pro Pro Arg Arg Arg Gln Leu Cys Phe Ser

2030 2035 2040

Arg Ile Val Arg Gly Pro Ala Asn Leu Arg Asn Leu Lys Glu Phe

2045 2050 2055

Lys Glu Glu Ile Leu Lys Gly Ala Gln Ser Glu Gly Lys Phe Leu

2060 2065 2070

Gly Asn Tyr Tyr Asn Glu Asp Lys Asp Lys Glu Lys Ala Leu Glu

2075 2080 2085

Ala Met Lys Asn Ser Phe Tyr Asp Tyr Glu Tyr Ile Ile Lys Gly

2090 2095 2100

Ser Asp Met Leu Thr Asn Ile Gln Phe Lys Asp Ile Lys Arg Lys

2105 2110 2115

Leu Asp Arg Leu Leu Glu Lys Glu Thr Asn Asn Thr Glu Lys Val

2120 2125 2130

Asp Asp Trp Trp Glu Thr Asn Lys Lys Ser Ile Trp Asn Ala Met

2135 2140 2145

Leu Cys Gly Tyr Lys Lys Ser Gly Asn Lys Ile Ile Asp Pro Ser

2150 2155 2160

Trp Cys Thr Ile Pro Thr Thr Glu Thr Pro Pro Gln Phe Leu Arg

2165 2170 2175

Trp Ile Lys Glu Trp Gly Thr Asn Val Cys Ile Gln Lys Glu Glu

2180 2185 2190

His Lys Glu Tyr Val Lys Ser Lys Cys Ser Asn Val Thr Asn Leu

2195 2200 2205

Gly Ala Gln Glu Ser Glu Ser Lys Asn Cys Thr Ser Glu Ile Lys

2210 2215 2220

Lys Tyr Gln Glu Trp Ser Arg Lys Arg Ser Ile Gln Trp Glu Ala

2225 2230 2235

Ile Ser Glu Gly Tyr Lys Lys Tyr Lys Gly Met Asp Glu Phe Lys

2240 2245 2250

Asn Thr Phe Lys Asn Ile Lys Glu Pro Asp Ala Asn Glu Pro Asn

2255 2260 2265

Ala Asn Glu Tyr Leu Lys Lys His Cys Ser Lys Cys Pro Cys Gly

2270 2275 2280

Phe Asn Asp Met Gln Glu Ile Thr Lys Tyr Thr Asn Ile Gly Asn

2285 2290 2295

Glu Ala Phe Lys Gln Ile Lys Glu Gln Val Asp Ile Pro Ala Glu

2300 2305 2310

Leu Glu Asp Val Ile Tyr Arg Leu Lys His His Glu Tyr Asp Lys

2315 2320 2325

Gly Asn Asp Tyr Ile Cys Asn Lys Tyr Lys Asn Ile Asn Val Asn

2330 2335 2340

Met Lys Lys Asn Asn Asp Asp Thr Trp Thr Asp Leu Val Lys Asn

2345 2350 2355

Ser Ser Asp Ile Asn Lys Gly Val Leu Leu Pro Pro Arg Arg Lys

2360 2365 2370

Asn Leu Phe Leu Lys Ile Asp Glu Ser Asp Ile Cys Lys Tyr Lys

2375 2380 2385

Arg Asp Pro Lys Leu Phe Lys Asp Phe Ile Tyr Ser Ser Ala Ile

2390 2395 2400

Ser Glu Val Glu Arg Leu Lys Lys Val Tyr Gly Glu Ala Lys Thr

2405 2410 2415

Lys Val Val His Ala Met Lys Tyr Ser Phe Ala Asp Ile Gly Ser

2420 2425 2430

Ile Ile Lys Gly Asp Asp Met Met Glu Asn Asn Ser Ser Asp Lys

2435 2440 2445

Ile Gly Lys Ile Leu Gly Asp Gly Val Gly Gln Asn Glu Lys Arg

2450 2455 2460

Lys Lys Trp Trp Asp Met Asn Lys Tyr His Ile Trp Glu Ser Met

2465 2470 2475

Leu Cys Gly Tyr Lys His Ala Tyr Gly Asn Ile Ser Glu Asn Asp

2480 2485 2490

Arg Lys Met Leu Asp Ile Pro Asn Asn Asp Asp Glu His Gln Phe

2495 2500 2505

Leu Arg Trp Phe Gln Glu Trp Thr Glu Asn Phe Cys Thr Lys Arg

2510 2515 2520

Asn Glu Leu Tyr Glu Asn Met Val Thr Ala Cys Asn Ser Ala Lys

2525 2530 2535

Cys Asn Thr Ser Asn Gly Ser Val Asp Lys Lys Glu Cys Thr Glu

2540 2545 2550

Ala Cys Lys Asn Tyr Ser Asn Phe Ile Leu Ile Lys Lys Lys Glu

2555 2560 2565

Tyr Gln Ser Leu Asn Ser Gln Tyr Asp Met Asn Tyr Lys Glu Thr

2570 2575 2580

Lys Ala Glu Lys Lys Glu Ser Pro Glu Tyr Phe Lys Asp Lys Cys

2585 2590 2595

Asn Gly Glu Cys Ser Cys Leu Ser Glu Tyr Phe Lys Asp Glu Thr

2600 2605 2610

Arg Trp Lys Asn Pro Tyr Glu Thr Leu Asp Asp Thr Glu Val Lys

2615 2620 2625

Asn Asn Cys Met Cys Lys Pro Pro Pro Pro Ala Ser Asn Ser Gly

2630 2635 2640

Arg Glu Leu

2645

<210> 4

<211> 7938

<212> DNA

<213> 人工合成序列

<400> 4

atggacaagt cctctatcgc caacaagatc gaagcctacc tgggagccaa gtctgacgac 60

tctaagatcg accagtctct gaaggccgac ccctctgaag tccagtacta cggatctggt 120

ggcgacggtt actacctgag gaagaacatc tgcaagatca ccgtcaacca ctctgactct 180

ggcaccaacg acccctgcga caggatcccc cctccctacg gcgacaacga ccagtggaag 240

tgcgccatca tcctgtctaa ggtgtccgaa aagcccgaaa acgtgttcgt cccccctagg 300

cgccagagga tgtgcatcaa caacctggaa aagctgaacg tcgacaagat cagggacaag 360

cacgccttcc tcgccgacgt cctgctgacc gctaggaacg aaggcgaaag gatcgtccag 420

aaccaccccg acaccaactc ttctaacgtc tgcaacgccc tggaacgctc tttcgccgac 480

atcgctgaca tcatccgcgg aaccgacctg tggaagggca ccaactctaa cctggaacag 540

aacctgaagc agatgttcgc caagatccgc gaaaacgaca aggtcctgca ggataagtac 600

cccaaggacc agaactaccg caagctgcgc gaggactggt ggaacgccaa caggcagaag 660

gtctgggaag tcatcacttg cggagccagg tctaacgacc tgctgatcaa gaggggatgg 720

cgcacctctg gaaagtctaa cggcgacaac aagctggaac tgtgccgcaa gtgcggacac 780

tacgaagaaa aggtccccac caagctggac tacgtccccc agttcctgcg ctggctgacc 840

gaatggatcg aggacttcta cagggaaaag cagaacctga tcgacgacat ggaaaggcac 900

agggaagaat gcacctctga ggaccacaag tctaaggaag gcacctctta ctgctctacc 960

tgcaaggaca agtgcaagaa gtactgcgag tgcgtcaaga agtggaagtc tgaatgggag 1020

aaccagaaga acaagtacac cgaactgtac cagcagaaca agaacgaaac ctctcagaag 1080

aacacctctc gctacgacga ctacgtcaag gacttcttca agaagctcga ggccaactac 1140

tcctccctgg aaaactacat caagggcgac ccctacttcg ccgaatacgc taccaagctg 1200

tccttcatcc tgaactcttc cgacgccaac aacccctctg agaagatcca aaagaacaac 1260

gacgaagtct gcaactgcaa cgaatctgga atcgcctctg tcgaacaaga gcagatctct 1320

gacccctcta gcaacaagac ctgcatcacc cactcttcta tcaaggccaa caagaagaag 1380

gtctgcaagc acgtcaagct gggagtcaga gagaacgaca aggacctccg cgtctgcgtc 1440

atcgaacaca cctccctgtc tggtgtcgaa aactgctgct gccaggactt cctgaggatc 1500

ctgcaagaaa actgctccga caacaagtcc ggatcttctt ctaacggctc ctgcaacaac 1560

aagaaccagg aagcctgcga gaagaacctc gaaaaggtcc tggcctctct gaccaactgc 1620

tacaagtgcg acaagtgcaa gtctgagcag agcaagaaga acaacaagaa ctggatctgg 1680

aagaagtcct ccggaaagga aggtggactg cagaaggaat acgccaacac catcggactg 1740

ccccccagga cccagtctct gtgcctggtc gtctgcctgg acgaaaaggg aaagaagacc 1800

caagagctga agaacatccg caccaactcc gaactgctga aggaatggat catcgccgcc 1860

ttccacgagg gaaagaacct caagccctct cacgaaaaga agaacgacga caacggcaag 1920

aagctgtgca aggccctcga gtactccttc gccgactacg gcgacctgat caagggaacc 1980

tctatctggg acaacgagta caccaaggac ttggaactga acctgcaaaa gatcttcggc 2040

aagctgttcc gcaagtacat caagaagaac aacaccgccg agcaggacac ctcctactct 2100

agcctggacg agctgaggga atcttggtgg aacactaaca agaagtacat ctggctggcc 2160

atgaagcacg gtgccggaat gaactctacc acttgctgcg gcgacggatc tgtgaccgga 2220

tctggatctt cttgcgacga catccccacc atcgacctga tccctcagta cctgcgcttc 2280

ctgcaagagt gggtcgaaca cttctgcaag cagaggcaag agaaggtcaa gcccgtcatc 2340

gagaactgca agtcttgcaa ggaaagcgga ggaacctgca acggcgaatg caagaccgag 2400

tgcaagaaca agtgtgaagt ctacaagaag ttcatcgagg actgcaaggg tggcgacgga 2460

accgccggat cttcctgggt caagcgctgg gaccagatct acaagaggta ctctaagtac 2520

atcgaggacg ccaagaggaa ccgcaaggcc ggcaccaaga actgcggacc ctcttctacc 2580

accaacgccg ccgaaaacaa gtgcgtccag tctgacatcg actcattctt caagcacctc 2640

atcgacatcg gactgaccac cccctcctct tacctgtcta tcgtcctgga cgacaacatc 2700

tgcggagccg acaaggcccc ctggaccacc tacaccactt acaccaccac cgaaaagtgc 2760

aacaaggaaa ccgacaagtc caagctccag cagtgcaaca ccgctgtcgt cgtcaacgtc 2820

ccctctcccc tgggaaacac cccccacgga tacaagtacg cctgccagtg caagatcccc 2880

accaacgaag aaacctgcga cgaccgcaag gagtacatga accagtggtc ttgcggatct 2940

gccaggacca tgaagagggg atacaagaac gacaactacg aactgtgcaa gtacaacggt 3000

gtcgacgtca agcccaccac cgtcaggtct aactcctcta agctggacga caaggacgtc 3060

accttcttca acctgttcga gcagtggaac aaggaaatcc agtaccagat cgagcagtac 3120

atgaccaaca ccaagatcag ctgcaacaac gaaaagaacg tcctgtctcg cgtcagcgac 3180

gaagccgccc agcccaagtt ctctgacaac gaaagggacc gcaactctat cacccacgag 3240

gacaagaact gcaaggaaaa gtgcaagtgc tactccctgt ggatcgagaa gatcaacgac 3300

caatgggaca agcagaagga caactacaac aagttccagc gcaagcagat ctacgacgct 3360

aacaagggat cccaaaacaa gaaggtcgtc agcctgtcca acttcctgtt cttcagctgc 3420

tgggaagagt acatccagaa gtacttcaac ggcgactggt ccaagatcaa gaacatcgga 3480

tctgacacct tcgagttcct catcaagaag tgcggaaacg actctggcga cggcgaaacc 3540

atcttctctg agaagctgaa caacgccgag aagaagtgca aggaaaacga gtccaccaac 3600

aacaagatga agtcctctga aacctcctgc gactgctctg aacccatcta catcagggga 3660

tgccagccca agatctacga cggaaagatc ttccccggaa agggcggaga aaagcagtgg 3720

atctgcaagg acaccatcat ccacggcgac accaacggtg cctgcatccc tcctaggacc 3780

cagaacctgt gcgtcggaga actgtgggac aagcgctacg gtggaaggtc taacatcaag 3840

aacgacacca aggaatccct gaagcagaag atcaagaacg ccatccagaa ggaaaccgaa 3900

ctgctgtacg agtaccacga caagggaacc gccatcatct ctaggaaccc catgaaggga 3960

cagaaggaaa aggaagagaa gaacaacgac tccaacggac tgcccaaggg attctgccac 4020

gccgtccagc gcagcttcat cgactacaag aacatgatcc tgggcacctc cgtcaacatc 4080

tacgagtaca tcggaaagct gcaagaggac atcaagaaga tcatcgagaa gggaaccacc 4140

aagcagaacg gaaagaccgt cggaagcgga gccgaaaacg tcaacgcctg gtggaaggga 4200

atcgaaggcg agatgtggga cgccgtccgc tgcgccatca ctaagatcaa caagaagcag 4260

aagaagaacg gcaccttctc tatcgacgag tgcggaatct tcccccccac cggaaacgac 4320

gaggaccagt ctgtctcctg gttcaaggaa tggtccgaac agttctgcat cgaaaggctg 4380

cagtacgaga agaacatcag ggacgcctgc accaacaacg gacagggcga caagatccaa 4440

ggcgactgca agcgcaagtg cgaagagtac aagaagtaca tcagcgagaa gaagcaagag 4500

tgggacaagc aaaagactaa gtacgagaac aagtacgtcg gaaagtctgc ctctgacctg 4560

ctcaaggaaa actaccccga atgcatctct gccaacttcg acttcatctt caacgacaac 4620

atcgagtaca agacctacta cccttacggc gactactctt ccatctgctc ttgcgaacaa 4680

gtgaagtact acgagtacaa caacgctgaa aagaagaaca acaagtccct gtgccacgag 4740

aagggaaacg accgcacctg gtctaagaag tacatcaaga agctcgagaa cggaaggacc 4800

ctggaaggcg tctacgtccc accccgcagg cagcagctgt gcctgtacga actgttcccc 4860

atcatcatca agaacaagaa cgacatcacc aacgccaaga aggaattgct ggaaaccctg 4920

cagatcgtcg ccgaaaggga agcctactac ctctggaagc agtaccacgc ccacaacgac 4980

accacctacc tggcccacaa gaaggcctgc tgcgctatca ggggatcttt ctacgacctc 5040

gaggatatca tcaagggcaa cgacctggtc cacgacgagt acaccaagta catcgactct 5100

aagctgaacg agatcttcga ctcctccaac aagaacgaca tcgaaactaa gagggccagg 5160

accgactggt gggaaaacga agctatcgcc gtccccaaca tcaccggtgc caacaagtct 5220

gaccccaaga ccatcaggca gctcgtctgg gacgccatgc agtctggtgt ccgcaaggcc 5280

atcgacgaag agaaggaaaa gaagaagccc aacgaaaact tcccaccctg catgggagtc 5340

cagcacatcg gaatcgccaa gccccagttc atccgttggc tggaagaatg gaccaacgag 5400

ttctgcgaga agtacactaa gtacttcgag gacatgaagt ccaactgcaa cctgcgcaag 5460

ggcgccgacg actgcgacga caactctaac atcgaatgca agaaggcttg cgccaactac 5520

accaactggc tgaaccccaa gaggatcgaa tggaacggaa tgtccaacta ctacaacaag 5580

atctaccgca agtccaacaa ggaatctgag gacggaaagg actactcaat gatcatggaa 5640

cccaccgtga tcgactacct gaacaagagg tgtaacggcg agatcaacgg caactacatc 5700

tgctgctctt gcaagaacat cggcgagaac tctacctctg gcaccgtgaa caagaagctg 5760

caaaagaagg aaactcagtg cgaggacaac aagggacccc tggacctgat gaacaaggtc 5820

ctcaacaaga tggaccccaa gtactctgag cacaagatga agtgcaccga ggtctacctg 5880

gaacacgtcg aagaacagtt gaaggaaatc gacaacgcca tcaaggacta caagctgtac 5940

cctctggacc gctgcttcga cgacaagagc aagatgaagg tttgcgacct catcggcgac 6000

gccatcggat gcaagcacaa gaccaagctc gacgaactgg acgagtggaa cgacgtcgac 6060

atgagggacc cttacaacaa gtacaagggc gtcctgatcc ccccaaggcg caggcagctg 6120

tgcttctcta ggatcgtcag gggacccgcc aacctgagga acctgaagga gttcaaggaa 6180

gagatcctga agggcgccca gtctgaggga aagttcctgg gaaactacta caacgaggac 6240

aaggacaagg aaaaggccct ggaagccatg aagaacagct tctacgacta cgagtacatc 6300

atcaagggct ctgacatgct gaccaacatc cagttcaagg acatcaagcg caagctggac 6360

aggctgctgg aaaaggaaac taacaacacc gaaaaggtgg acgactggtg ggagactaac 6420

aagaagagta tctggaacgc catgttgtgc ggttacaaga agtccggcaa caagatcatc 6480

gacccctctt ggtgcaccat ccctaccacc gaaacccccc ctcagttctt gcgttggatc 6540

aaggaatggg gcaccaacgt ctgcatccaa aaggaagaac acaaggaata cgtcaagagc 6600

aagtgctcca acgtcaccaa cctcggagcc caagaatctg aatctaagaa ctgcacctcc 6660

gagatcaaga agtaccaaga gtggtcccgc aagcgctcta tccagtggga agccatctct 6720

gagggctaca agaagtacaa gggtatggac gagttcaaga acaccttcaa gaacatcaag 6780

gaacccgacg ccaacgagcc caacgccaac gaatacctga agaagcactg ctctaagtgc 6840

ccctgcggat tcaacgacat gcaagaaatc actaagtaca ccaacatcgg caacgaggcc 6900

ttcaagcaga tcaaggaaca ggtcgacatc cccgccgaat tggaggacgt catctacagg 6960

ctgaagcacc acgaatacga caagggaaac gactacatct gcaacaagta caagaacatc 7020

aacgtcaaca tgaagaagaa caacgacgac acctggaccg acctggtcaa gaactcttct 7080

gacatcaaca agggtgtcct gctgccccct cgccgcaaga acctgttcct gaagatcgac 7140

gaatccgaca tctgcaagta caagagggac cctaagctgt tcaaggactt catctactct 7200

tccgccatct ccgaagtcga acgcctcaag aaggtctacg gcgaggccaa gaccaaggtc 7260

gtccacgcta tgaagtactc attcgcagac atcggctcca tcatcaaggg cgacgacatg 7320

atggaaaaca acagctctga caagatcggc aagatcctgg gcgacggtgt cggccagaac 7380

gaaaagcgca agaagtggtg ggacatgaac aagtaccaca tctgggagtc tatgctgtgc 7440

ggctacaagc acgcttacgg aaacatctct gagaacgacc gaaagatgct ggacatcccc 7500

aacaacgacg acgaacacca gttcctccgc tggttccaag aatggactga aaacttctgc 7560

accaagcgca acgagctgta cgaaaacatg gtcaccgcct gcaactctgc caagtgcaac 7620

acttctaacg gatccgtgga caagaaggaa tgcaccgaag cctgcaagaa ctactccaac 7680

ttcatcctga tcaagaagaa ggaataccag tccctgaact cccagtacga catgaactac 7740

aaggaaacta aggccgaaaa gaaggaatcc cccgagtact tcaaggacaa gtgtaacggc 7800

gaatgctctt gcctgagcga atacttcaag gacgaaacca ggtggaagaa cccctacgaa 7860

accctcgacg acaccgaagt caagaacaac tgcatgtgca agcccccacc ccctgcctct 7920

aacagcggcc gcgagctc 7938

<210> 5

<211> 2649

<212> PRT

<213> 人工合成序列

<400> 5

Met Asp Ser Thr Ser Thr Ile Ala Asn Lys Ile Glu Glu Tyr Leu Gly

1 5 10 15

Ala Lys Ser Asp Asp Ser Lys Ile Asp Glu Leu Leu Lys Ala Asp Pro

20 25 30

Ser Glu Val Glu Tyr Tyr Arg Ser Gly Gly Asp Gly Asp Tyr Leu Lys

35 40 45

Asn Asn Ile Cys Lys Ile Thr Val Asn His Ser Asp Ser Gly Lys Tyr

50 55 60

Asp Pro Cys Glu Lys Lys Leu Pro Pro Tyr Asp Asp Asn Asp Gln Trp

65 70 75 80

Lys Cys Gln Gln Asn Ser Ser Asp Gly Ser Gly Lys Pro Glu Asn Ile

85 90 95

Cys Val Pro Pro Arg Arg Glu Arg Leu Cys Thr Tyr Asn Leu Glu Asn

100 105 110

Leu Lys Phe Asp Lys Ile Arg Asp Asn Asn Ala Phe Leu Ala Asp Val

115 120 125

Leu Leu Thr Ala Arg Asn Glu Gly Glu Lys Ile Val Gln Asn His Pro

130 135 140

Asp Thr Asn Ser Ser Asn Val Cys Asn Ala Leu Glu Arg Ser Phe Ala

145 150 155 160

Asp Leu Ala Asp Ile Ile Arg Gly Thr Asp Gln Trp Lys Gly Thr Asn

165 170 175

Ser Asn Leu Glu Lys Asn Leu Lys Gln Met Phe Ala Lys Ile Arg Glu

180 185 190

Asn Asp Lys Val Leu Gln Asp Lys Tyr Pro Lys Asp Gln Lys Tyr Thr

195 200 205

Lys Leu Arg Glu Ala Trp Trp Asn Ala Asn Arg Gln Lys Val Trp Glu

210 215 220

Val Ile Thr Cys Gly Ala Arg Ser Asn Asp Leu Leu Ile Lys Arg Gly

225 230 235 240

Trp Arg Thr Ser Gly Lys Ser Asp Arg Lys Lys Asn Phe Glu Leu Cys

245 250 255

Arg Lys Cys Gly His Tyr Glu Lys Glu Val Pro Thr Lys Leu Asp Tyr

260 265 270

Val Pro Gln Phe Leu Arg Trp Leu Thr Glu Trp Ile Glu Asp Phe Tyr

275 280 285

Arg Glu Lys Gln Asn Leu Ile Asp Asp Met Glu Arg His Arg Glu Glu

290 295 300

Cys Thr Arg Glu Asp His Lys Ser Lys Glu Gly Thr Ser Tyr Cys Ser

305 310 315 320

Thr Cys Lys Asp Lys Cys Lys Lys Tyr Cys Glu Cys Val Lys Lys Trp

325 330 335

Lys Thr Glu Trp Glu Asn Gln Glu Asn Lys Tyr Lys Asp Leu Tyr Glu

340 345 350

Gln Asn Lys Asn Lys Thr Ser Gln Lys Asn Thr Ser Arg Tyr Asp Asp

355 360 365

Tyr Val Lys Asp Phe Phe Glu Lys Leu Glu Ala Asn Tyr Ser Ser Leu

370 375 380

Glu Asn Tyr Ile Lys Gly Asp Pro Tyr Phe Ala Glu Tyr Ala Thr Lys

385 390 395 400

Leu Ser Phe Ile Leu Asn Pro Ser Asp Ala Asn Asn Pro Ser Gly Glu

405 410 415

Thr Ala Asn His Asn Asp Glu Ala Cys Asn Cys Asn Glu Ser Gly Ile

420 425 430

Ser Ser Val Gly Gln Ala Gln Thr Ser Gly Pro Ser Ser Asn Lys Thr

435 440 445

Cys Ile Thr His Ser Ser Ile Lys Thr Asn Lys Lys Lys Glu Cys Lys

450 455 460

Asp Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Lys Ile Cys

465 470 475 480

Val Ile Glu Asp Thr Ser Leu Ser Gly Val Asp Asn Cys Cys Cys Gln

485 490 495

Asp Leu Leu Gly Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Arg Gly

500 505 510

Ser Ser Ser Asn Asp Ser Cys Asp Asn Lys Asn Gln Asp Glu Cys Gln

515 520 525

Lys Lys Leu Glu Lys Val Phe Ala Ser Leu Thr Asn Gly Tyr Lys Cys

530 535 540

Asp Lys Cys Lys Ser Gly Thr Ser Arg Ser Lys Lys Lys Trp Ile Trp

545 550 555 560

Lys Lys Ser Ser Gly Asn Glu Glu Gly Leu Gln Glu Glu Tyr Ala Asn

565 570 575

Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Tyr Leu Gly Asn Leu

580 585 590

Pro Lys Leu Glu Asn Val Cys Glu Asp Val Lys Asp Ile Asn Phe Asp

595 600 605

Thr Lys Glu Lys Phe Leu Ala Gly Cys Leu Ile Val Ser Phe His Glu

610 615 620

Gly Lys Asn Leu Lys Lys Arg Tyr Pro Gln Asn Lys Asn Ser Gly Asn

625 630 635 640

Lys Glu Asn Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr Gly

645 650 655

Asp Leu Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys Asp

660 665 670

Leu Glu Leu Asn Leu Gln Asn Asn Phe Gly Lys Leu Phe Gly Lys Tyr

675 680 685

Ile Lys Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser Leu

690 695 700

Asp Glu Leu Arg Glu Ser Trp Trp Asn Thr Asn Lys Lys Tyr Ile Trp

705 710 715 720

Thr Ala Met Lys His Gly Ala Glu Met Asn Ile Thr Thr Cys Asn Ala

725 730 735

Asp Gly Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro Thr

740 745 750

Ile Asp Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val Glu

755 760 765

Asn Phe Cys Glu Gln Arg Gln Ala Lys Val Lys Asp Val Ile Thr Asn

770 775 780

Cys Lys Ser Cys Lys Glu Ser Gly Asn Lys Cys Lys Thr Glu Cys Lys

785 790 795 800

Thr Lys Cys Lys Asp Glu Cys Glu Lys Tyr Lys Lys Phe Ile Glu Ala

805 810 815

Cys Gly Thr Ala Gly Gly Gly Ile Gly Thr Ala Gly Ser Pro Trp Ser

820 825 830

Lys Arg Trp Asp Gln Ile Tyr Lys Arg Tyr Ser Lys His Ile Glu Asp

835 840 845

Ala Lys Arg Asn Arg Lys Ala Gly Thr Lys Asn Cys Gly Thr Ser Ser

850 855 860

Thr Thr Asn Ala Ala Ala Ser Thr Asp Glu Asn Lys Cys Val Gln Ser

865 870 875 880

Asp Ile Asp Ser Phe Phe Lys His Leu Ile Asp Ile Gly Leu Thr Thr

885 890 895

Pro Ser Ser Tyr Leu Ser Asn Val Leu Asp Asp Asn Ile Cys Gly Ala

900 905 910

Asp Lys Ala Pro Trp Thr Thr Tyr Thr Thr Tyr Thr Thr Thr Glu Lys

915 920 925

Cys Asn Lys Glu Arg Asp Lys Ser Lys Ser Gln Ser Ser Asp Thr Leu

930 935 940

Val Val Val Asn Val Pro Ser Pro Leu Gly Asn Thr Pro Tyr Arg Tyr

945 950 955 960

Lys Tyr Ala Cys Gln Cys Lys Ile Pro Thr Asn Glu Glu Thr Cys Asp

965 970 975

Asp Arg Lys Glu Tyr Met Asn Gln Trp Ser Cys Gly Ser Ala Arg Thr

980 985 990

Met Lys Arg Gly Tyr Lys Asn Asp Asn Tyr Glu Leu Cys Lys Tyr Asn

995 1000 1005

Gly Val Asp Val Lys Pro Thr Thr Val Arg Ser Asn Ser Ser Lys

1010 1015 1020

Leu Asp Gly Asn Asp Val Thr Phe Phe Asn Leu Phe Glu Gln Trp

1025 1030 1035

Asn Lys Glu Ile Gln Tyr Gln Ile Glu Gln Tyr Met Thr Asn Ala

1040 1045 1050

Asn Ile Ser Cys Ile Asp Glu Lys Glu Val Leu Asp Ser Val Ser

1055 1060 1065

Asp Glu Gly Thr Pro Lys Val Arg Gly Gly Tyr Glu Asp Gly Arg

1070 1075 1080

Asn Asn Asn Thr Asp Gln Gly Thr Asn Cys Lys Glu Lys Cys Lys

1085 1090 1095

Cys Tyr Lys Leu Trp Ile Glu Lys Ile Asn Asp Gln Trp Gly Lys

1100 1105 1110

Gln Lys Asp Asn Tyr Asn Lys Phe Arg Ser Lys Gln Ile Tyr Asp

1115 1120 1125

Ala Asn Lys Gly Ser Gln Asn Lys Lys Val Val Ser Leu Ser Asn

1130 1135 1140

Phe Leu Phe Phe Ser Cys Trp Glu Glu Tyr Ile Gln Lys Tyr Phe

1145 1150 1155

Asn Gly Asp Trp Ser Lys Ile Lys Asn Ile Gly Ser Asp Thr Phe

1160 1165 1170

Glu Phe Leu Ile Lys Lys Cys Gly Asn Asn Ser Ala His Gly Glu

1175 1180 1185

Glu Ile Phe Ser Glu Lys Leu Lys Asn Ala Glu Lys Lys Cys Lys

1190 1195 1200

Glu Asn Glu Ser Thr Asp Thr Asn Ile Asn Lys Ser Glu Thr Ser

1205 1210 1215

Cys Asp Leu Asn Ala Thr Asn Tyr Ile Arg Gly Cys Gln Ser Lys

1220 1225 1230

Thr Tyr Asp Gly Lys Ile Phe Pro Gly Lys Gly Gly Glu Lys Gln

1235 1240 1245

Trp Ile Cys Lys Asp Thr Ile Ile His Gly Asp Thr Asn Gly Ala

1250 1255 1260

Cys Ile Pro Pro Arg Thr Gln Asn Leu Cys Val Gly Glu Leu Trp

1265 1270 1275

Asp Lys Ser Tyr Gly Gly Arg Ser Asn Ile Lys Asn Asp Thr Lys

1280 1285 1290

Glu Leu Leu Lys Glu Lys Ile Lys Asn Ala Ile His Lys Glu Thr

1295 1300 1305

Glu Leu Leu Tyr Glu Tyr His Asp Thr Gly Thr Ala Ile Ile Ser

1310 1315 1320

Lys Asn Asp Lys Lys Gly Gln Lys Gly Lys Asn Asp Pro Asn Gly

1325 1330 1335

Leu Pro Lys Gly Phe Cys His Ala Val Gln Arg Ser Phe Ile Asp

1340 1345 1350

Tyr Lys Asn Met Ile Leu Gly Thr Ser Val Asn Ile Tyr Glu His

1355 1360 1365

Ile Gly Lys Leu Gln Glu Asp Ile Lys Lys Ile Ile Glu Lys Gly

1370 1375 1380

Thr Pro Gln Gln Lys Asp Lys Ile Gly Gly Val Gly Ser Ser Thr

1385 1390 1395

Glu Asn Val Asn Ala Trp Trp Lys Gly Ile Glu Arg Glu Met Trp

1400 1405 1410

Asp Ala Val Arg Cys Ala Ile Thr Lys Ile Asn Lys Lys Asn Asn

1415 1420 1425

Asn Ser Ile Phe Asn Gly Asp Glu Cys Gly Val Ser Pro Pro Thr

1430 1435 1440

Gly Asn Asp Glu Asp Gln Ser Val Ser Trp Phe Lys Glu Trp Gly

1445 1450 1455

Glu Gln Phe Cys Ile Glu Arg Leu Arg Tyr Glu Gln Asn Ile Arg

1460 1465 1470

Glu Ala Cys Thr Ile Asn Gly Lys Asn Glu Lys Lys Cys Ile Asn

1475 1480 1485

Ser Lys Ser Gly Gln Gly Asp Lys Ile Gln Gly Ala Cys Lys Arg

1490 1495 1500

Lys Cys Glu Lys Tyr Lys Lys Tyr Ile Ser Glu Lys Lys Gln Glu

1505 1510 1515

Trp Asp Lys Gln Lys Thr Lys Tyr Glu Asn Lys Tyr Val Gly Lys

1520 1525 1530

Ser Ala Ser Asp Leu Leu Lys Glu Asn Tyr Pro Glu Cys Ile Ser

1535 1540 1545

Ala Asn Phe Asp Phe Ile Phe Asn Asp Asn Ile Glu Tyr Lys Thr

1550 1555 1560

Tyr Tyr Pro Tyr Gly Asp Tyr Ser Ser Ile Cys Ser Cys Glu Gln

1565 1570 1575

Val Lys Tyr Tyr Lys Tyr Asn Asn Ala Glu Lys Lys Asn Asn Lys

1580 1585 1590

Ser Leu Cys Tyr Glu Lys Asp Asn Asp Met Thr Trp Ser Lys Lys

1595 1600 1605

Tyr Ile Lys Lys Leu Glu Asn Gly Arg Ser Leu Glu Gly Val Tyr

1610 1615 1620

Val Pro Pro Arg Arg Gln Gln Leu Cys Leu Tyr Glu Leu Phe Pro

1625 1630 1635

Ile Ile Ile Lys Asn Glu Glu Gly Met Glu Lys Ala Lys Glu Glu

1640 1645 1650

Leu Leu Glu Thr Leu Gln Ile Val Ala Glu Arg Glu Ala Tyr Tyr

1655 1660 1665

Leu Trp Lys Gln Tyr Asn Pro Thr Gly Lys Gly Ile Asp Asp Ala

1670 1675 1680

Asn Lys Lys Ala Cys Cys Ala Ile Arg Gly Ser Phe Tyr Asp Leu

1685 1690 1695

Glu Asp Ile Ile Lys Gly Asn Asp Leu Val His Asp Glu Tyr Thr

1700 1705 1710

Lys Tyr Ile Asp Ser Lys Leu Asn Glu Ile Phe Gly Ser Ser Asn

1715 1720 1725

Thr Asn Asp Ile Asp Thr Lys Arg Ala Arg Thr Asp Trp Trp Glu

1730 1735 1740

Asn Glu Thr Ile Thr Asn Gly Thr Asp Arg Lys Thr Ile Arg Gln

1745 1750 1755

Leu Val Trp Asp Ala Met Gln Ser Gly Val Arg Tyr Ala Val Glu

1760 1765 1770

Glu Lys Asn Glu Asn Phe Pro Leu Cys Met Gly Val Glu His Ile

1775 1780 1785

Gly Ile Ala Lys Pro Gln Phe Ile Arg Trp Leu Glu Glu Trp Thr

1790 1795 1800

Asn Glu Phe Cys Glu Lys Tyr Thr Lys Tyr Phe Glu Asp Met Lys

1805 1810 1815

Ser Lys Cys Asp Pro Pro Lys Arg Ala Asp Thr Cys Gly Asp Asn

1820 1825 1830

Ser Asn Ile Glu Cys Lys Lys Ala Cys Ala Asn Tyr Thr Asn Trp

1835 1840 1845

Leu Asn Pro Lys Arg Ile Glu Trp Asn Gly Met Ser Asn Tyr Tyr

1850 1855 1860

Asn Lys Ile Tyr Arg Lys Ser Asn Lys Glu Ser Glu Asp Gly Lys

1865 1870 1875

Asp Tyr Ser Met Ile Met Ala Pro Thr Val Ile Asp Tyr Leu Asn

1880 1885 1890

Lys Arg Cys His Gly Glu Ile Asn Gly Asn Tyr Ile Cys Cys Ser

1895 1900 1905

Cys Lys Asn Ile Gly Ala Tyr Asn Thr Thr Ser Gly Thr Val Asn

1910 1915 1920

Lys Lys Leu Gln Lys Lys Glu Thr Glu Cys Glu Glu Glu Lys Gly

1925 1930 1935

Pro Leu Asp Leu Met Asn Glu Val Leu Asn Lys Met Asp Lys Lys

1940 1945 1950

Tyr Ser Ala His Lys Met Lys Cys Thr Glu Val Tyr Leu Glu His

1955 1960 1965

Val Glu Glu Gln Leu Asn Glu Ile Asp Asn Ala Ile Lys Asp Tyr

1970 1975 1980

Lys Leu Tyr Pro Leu Asp Arg Cys Phe Asp Asp Gln Thr Lys Met

1985 1990 1995

Lys Val Cys Asp Leu Ile Ala Asp Ala Ile Gly Cys Lys Asp Lys

2000 2005 2010

Thr Lys Leu Asp Glu Leu Asp Glu Trp Asn Asp Met Asp Leu Arg

2015 2020 2025

Gly Thr Tyr Asn Lys His Lys Gly Val Leu Ile Pro Pro Arg Arg

2030 2035 2040

Arg Gln Leu Cys Phe Ser Arg Ile Val Arg Gly Pro Ala Asn Leu

2045 2050 2055

Arg Ser Leu Asn Glu Phe Lys Glu Glu Ile Leu Lys Gly Ala Gln

2060 2065 2070

Ser Glu Gly Lys Phe Leu Gly Asn Tyr Tyr Lys Glu His Lys Asp

2075 2080 2085

Lys Glu Lys Ala Leu Glu Ala Met Lys Asn Ser Phe Tyr Asp Tyr

2090 2095 2100

Glu Asp Ile Ile Lys Gly Thr Asp Met Leu Thr Asn Ile Glu Phe

2105 2110 2115

Lys Asp Ile Lys Ile Lys Leu Asp Arg Leu Leu Glu Lys Glu Thr

2120 2125 2130

Asn Asn Thr Lys Lys Ala Glu Asp Trp Trp Lys Thr Asn Lys Lys

2135 2140 2145

Ser Ile Trp Asn Ala Met Leu Cys Gly Tyr Lys Lys Ser Gly Asn

2150 2155 2160

Lys Ile Ile Asp Pro Ser Trp Cys Thr Ile Pro Thr Thr Glu Thr

2165 2170 2175

Pro Pro Gln Phe Leu Arg Trp Ile Lys Glu Trp Gly Thr Asn Val

2180 2185 2190

Cys Ile Gln Lys Gln Glu His Lys Glu Tyr Val Lys Ser Lys Cys

2195 2200 2205

Ser Asn Val Thr Asn Leu Gly Ala Gln Ala Ser Glu Ser Asn Asn

2210 2215 2220

Cys Thr Ser Glu Ile Lys Lys Tyr Gln Glu Trp Ser Arg Lys Arg

2225 2230 2235

Ser Ile Gln Trp Glu Thr Ile Ser Lys Arg Tyr Lys Lys Tyr Lys

2240 2245 2250

Arg Met Asp Ile Leu Lys Asp Val Lys Glu Pro Asp Ala Asn Thr

2255 2260 2265

Tyr Leu Arg Glu His Cys Ser Lys Cys Pro Cys Gly Phe Asn Asp

2270 2275 2280

Met Glu Glu Met Asn Asn Asn Glu Asp Asn Glu Lys Glu Ala Phe

2285 2290 2295

Lys Gln Ile Lys Glu Gln Val Lys Ile Pro Ala Glu Leu Glu Asp

2300 2305 2310

Val Ile Tyr Arg Ile Lys His His Glu Tyr Asp Lys Gly Asn Asp

2315 2320 2325

Tyr Ile Cys Asn Lys Tyr Lys Asn Ile His Asp Arg Met Lys Lys

2330 2335 2340

Asn Asn Gly Asn Phe Val Thr Asp Asn Phe Val Lys Lys Ser Trp

2345 2350 2355

Glu Ile Ser Asn Gly Val Leu Ile Pro Pro Arg Arg Lys Asn Leu

2360 2365 2370

Phe Leu Tyr Ile Asp Pro Ser Lys Ile Cys Glu Tyr Lys Lys Asp

2375 2380 2385

Pro Lys Leu Phe Lys Asp Phe Ile Tyr Trp Ser Ala Phe Thr Glu

2390 2395 2400

Val Glu Arg Leu Lys Lys Ala Tyr Gly Gly Ala Arg Ala Lys Val

2405 2410 2415

Val His Ala Met Lys Tyr Ser Phe Thr Asp Ile Gly Ser Ile Ile

2420 2425 2430

Lys Gly Asp Asp Met Met Glu Lys Asn Ser Ser Asp Lys Ile Gly

2435 2440 2445

Lys Ile Leu Gly Asp Thr Asp Gly Gln Asn Glu Lys Arg Lys Lys

2450 2455 2460

Trp Trp Asp Met Asn Lys Tyr His Ile Trp Glu Ser Met Leu Cys

2465 2470 2475

Gly Tyr Arg Glu Ala Glu Gly Asp Thr Glu Thr Asn Glu Asn Cys

2480 2485 2490

Arg Phe Pro Asp Ile Glu Ser Val Pro Gln Phe Leu Arg Trp Phe

2495 2500 2505

Gln Glu Trp Ser Glu Asn Phe Cys Asp Arg Arg Gln Lys Leu Tyr

2510 2515 2520

Asp Lys Leu Asn Ser Glu Cys Ile Ser Ala Glu Cys Thr Asn Gly

2525 2530 2535

Ser Val Asp Asn Ser Lys Cys Thr His Ala Cys Val Asn Tyr Lys

2540 2545 2550

Asn Tyr Ile Leu Thr Lys Lys Thr Glu Tyr Glu Ile Gln Thr Asn

2555 2560 2565

Lys Tyr Asp Asn Glu Phe Lys Asn Lys Asn Ser Asn Asp Lys Asp

2570 2575 2580

Ala Pro Asp Tyr Leu Lys Glu Lys Cys Asn Asp Asn Lys Cys Glu

2585 2590 2595

Cys Leu Asn Lys His Ile Asp Asp Lys Asn Lys Thr Trp Lys Asn

2600 2605 2610

Pro Tyr Glu Thr Leu Glu Asp Thr Phe Lys Ser Lys Cys Asp Cys

2615 2620 2625

Pro Lys Pro Leu Pro Ser Pro Ile Lys Pro Asp Asp Leu Pro Pro

2630 2635 2640

Gln Ala Asp Glu Pro Phe

2645

<210> 6

<211> 7947

<212> DNA

<213> 人工合成序列

<400> 6

atggacagca ccagcacaat cgccaacaag atcgaggaat atctgggagc taagtccgat 60

gacagcaaga tcgacgaact gctgaaggcc gatcctagcg aagtggagta ctacagaagc 120

ggaggcgacg gcgactacct gaagaacaac atctgcaaga tcaccgtgaa ccacagcgat 180

agcggcaagt atgacccctg cgagaagaag ctgcccccct acgacgacaa cgaccagtgg 240

aagtgccagc agaacagcag cgacggcagc ggcaagcccg agaacatctg cgtgcccccc 300

agacgggagc ggctgtgcac ctacaacctg gaaaacctga agttcgacaa gatccgggac 360

aacaacgcct tcctggccga cgtgctgctg accgcccgga acgagggcga gaagatcgtg 420

cagaaccacc ccgacaccaa cagcagcaac gtgtgcaacg ccctggaacg gtccttcgct 480

gacctggctg acatcatccg gggcaccgat cagtggaagg gcaccaactc caatctggaa 540

aagaacctga agcagatgtt cgccaagatc agagaaaacg acaaggtgct gcaggacaag 600

taccccaagg accagaagta caccaagctg cgggaggcct ggtggaacgc caaccggcag 660

aaagtgtggg aagtgatcac ctgtggcgcc agaagcaacg atctgctgat caagcggggc 720

tggcggacca gcggcaagag cgaccggaag aaaaacttcg agctgtgccg gaagtgcggc 780

cactacgaga aagaggtgcc caccaagctg gactacgtgc cccagttcct gcggtggctg 840

accgagtgga tcgaggactt ctaccgggag aagcagaacc tgatcgacga catggaacgg 900

caccgggagg aatgcaccag agaggaccac aagagcaaag agggcaccag ctactgcagc 960

acatgcaagg acaagtgcaa gaaatactgc gagtgcgtga agaaatggaa aaccgagtgg 1020

gagaaccagg aaaacaagta caaggacctg tacgagcaga acaagaacaa gaccagccag 1080

aagaacacca gcagatacga cgactacgtg aaggacttct tcgagaagct ggaagccaac 1140

tacagcagcc tggaaaacta catcaagggc gacccctatt tcgctgagta cgctacaaaa 1200

ctgagcttca tcctgaaccc cagcgacgcc aacaacccca gcggcgagac agccaaccac 1260

aacgacgagg cctgcaactg caacgagagc ggcatcagca gcgtgggcca ggctcagaca 1320

tccggcccta gcagcaacaa gacctgtatc acccacagct ccatcaagac caacaagaaa 1380

aaagaatgca aggacgtgaa gctgggcgtg cgggagaacg acaaggatct gaagatctgc 1440

gtgatcgagg acaccagcct gagcggcgtg gacaactgct gctgccagga tctgctgggc 1500

atcctgcagg aaaactgcag cgacaacaag cggggcagca gctccaacga cagctgcgac 1560

aataagaacc aggacgagtg ccagaaaaag ctggaaaagg tgttcgccag cctgaccaac 1620

ggctacaagt gcgataagtg caagagcggc acctcccggt ccaagaagaa gtggatctgg 1680

aagaagtcca gcggcaacga ggaaggcctg caggaagagt acgccaacac catcggcctg 1740

ccccccagga cccagagcct gtacctgggc aatctgccca aactggaaaa cgtgtgcgag 1800

gatgtgaagg acatcaactt cgacaccaaa gagaagtttc tggccggctg cctgatcgtg 1860

tccttccacg agggcaagaa tctgaagaag cgctaccccc agaataagaa cagcggcaac 1920

aaagaaaacc tgtgcaaggc tctggaatac agcttcgccg actacggcga cctgatcaag 1980

ggcacctcca tctgggacaa cgagtacaca aaggacctgg aactgaatct gcagaacaac 2040

ttcggcaagc tgttcggcaa gtacatcaag aagaacaata ccgccgagca ggacacctcc 2100

tacagctccc tggacgagct gcgcgagtct tggtggaata ccaataagaa gtacatctgg 2160

accgccatga agcacggcgc cgagatgaac atcaccacct gtaacgccga cggctccgtg 2220

accggcagcg gctccagctg cgacgacatc cccaccatcg acctgatccc ccagtacctg 2280

agatttctgc aggaatgggt cgagaacttc tgcgagcagc ggcaggccaa agtgaaggac 2340

gtgatcacca actgcaagag ctgcaaagaa tccggcaaca aatgcaagac cgagtgcaaa 2400

accaagtgca aggatgagtg cgagaagtac aagaagttca tcgaggcctg cggcacagcc 2460

ggcggaggca tcggaacagc cggcagcccc tggtccaaga gatgggacca gatctacaag 2520

cggtacagca agcacatcga ggacgccaag cggaaccgga aggccggcac caagaactgc 2580

ggcaccagct ccaccaccaa cgccgctgcc agcaccgacg agaataagtg cgtgcagagc 2640

gacatcgaca gctttttcaa gcacctgatc gatatcggcc tgaccacccc cagcagctac 2700

ctgagcaacg tgctggacga caacatctgt ggcgccgaca aggccccctg gacaacctat 2760

acaacataca ctacaaccga gaagtgcaac aaagagcggg acaagagcaa gagccagagc 2820

agcgacaccc tggtggtggt gaacgtgccc agccccctgg gcaacacacc ctaccggtac 2880

aagtacgcct gccagtgcaa gatccccacc aacgaggaaa catgcgacga ccggaaagaa 2940

tacatgaacc agtggtcctg cgggagcgct cggaccatga agagagggta taagaacgat 3000

aactacgaac tgtgcaagta caacggcgtg gatgtgaagc ccaccaccgt gcggagcaac 3060

tccagcaagc tggacggcaa cgacgtgacc ttcttcaacc tgttcgagca gtggaacaaa 3120

gaaatccagt accagatcga gcagtacatg accaacgcca acatcagctg catcgatgag 3180

aaagaagtgc tggacagcgt ctccgacgag ggcaccccca aagtgcgggg aggctacgag 3240

gacggccgga acaacaacac agatcagggc acaaattgca aagaaaagtg caagtgctac 3300

aagctgtgga tcgagaagat caacgatcag tggggcaagc agaaggacaa ctacaacaag 3360

ttccggtcca agcagatcta cgacgccaat aagggcagcc agaacaaaaa ggtggtgtcc 3420

ctgagcaact tcctgttctt cagctgctgg gaggaataca tccagaagta cttcaacggc 3480

gattggagca agatcaagaa catcggctcc gacaccttcg agtttctgat caagaagtgc 3540

ggcaacaaca gcgcccacgg cgaggaaatc ttcagcgaga agctgaagaa cgccgagaag 3600

aagtgcaaag agaacgagag caccgacacc aatatcaaca agagcgagac atcctgcgac 3660

ctgaacgcca ccaactacat ccggggctgc cagagcaaga cctacgacgg caagatcttc 3720

cccggcaagg gcggcgagaa gcagtggatt tgcaaggaca ccatcatcca cggcgacacc 3780

aacggcgcct gcatcccccc tcgcacccag aacctgtgcg tgggcgagct gtgggacaag 3840

tcctacggcg gcagatccaa catcaagaac gataccaaag aactgctgaa agagaagatt 3900

aagaacgcca tccacaaaga gacagagctg ctgtacgagt accacgacac cggcaccgcc 3960

atcatcagca agaatgacaa gaagggccag aagggcaaga acgaccctaa cggcctgccc 4020

aaaggcttct gtcatgctgt gcagcggagc ttcatcgact acaagaacat gatcctgggc 4080

accagcgtga acatctacga gcacatcggc aagctgcagg aagatatcaa gaagatcatc 4140

gaaaagggca cccctcagca gaaggataag atcggcggcg tgggcagcag caccgagaac 4200

gtgaacgctt ggtggaaggg aatcgagcgg gagatgtggg acgccgtgag atgcgccatc 4260

accaagatca ataagaagaa taacaacagc atcttcaatg gcgacgagtg cggcgtgagc 4320

ccccccaccg gcaacgatga ggaccagagc gtgtcctggt tcaaagagtg gggcgagcag 4380

ttctgcatcg agcggctgag atacgaacag aacatcagag aggcctgcac catcaacggc 4440

aaaaacgaga agaaatgcat caacagcaag tccggccagg gcgataagat ccagggcgcc 4500

tgcaagcgga agtgtgaaaa gtataagaag tatatcagcg agaaaaaaca ggaatgggac 4560

aagcagaaaa ccaagtacga gaacaaatac gtgggcaaga gcgccagcga tctgctgaaa 4620

gaaaactacc ccgagtgcat cagcgccaac ttcgacttca tcttcaacga caatatcgag 4680

tacaagacct actaccccta cggcgattac agcagcatct gcagctgcga acaggtgaag 4740

tactataagt acaacaatgc cgagaaaaag aacaacaaga gcctgtgcta cgagaaggac 4800

aatgacatga cctggtctaa gaaatatatc aagaagctgg aaaacggccg gtccctggaa 4860

ggcgtgtacg tgccccctcg gcggcagcag ctgtgtctgt acgagctgtt ccctatcatc 4920

atcaaaaacg aagagggcat ggaaaaggcc aaagaggaac tgctggaaac cctgcagatc 4980

gtggccgagc gcgaggccta ctacctgtgg aagcagtaca accccacagg caaagggatt 5040

gatgatgcca acaagaaggc ctgctgcgcc atcagaggca gcttctacga cctggaagat 5100

attatcaagg gcaacgacct ggtgcacgac gagtacacca aatacatcga ctccaagctg 5160

aacgagatct tcggctccag caacaccaac gacatcgata ccaagcgggc cagaaccgat 5220

tggtgggaga acgagacaat caccaacggc accgacagaa agaccatccg gcagctggtg 5280

tgggatgcca tgcagagcgg agtgcgatat gctgtggagg aaaagaacga gaacttcccc 5340

ctgtgcatgg gcgtggagca cattggcatt gccaagcccc agttcatcag atggctggaa 5400

gagtggacca acgagttctg cgagaaatac accaagtact tcgaggacat gaagtccaag 5460

tgcgaccccc ccaagagggc cgacacatgc ggcgacaact ccaacatcga gtgcaagaag 5520

gcttgcgcca actacaccaa ctggctgaac cccaagcgga tcgagtggaa cggcatgagc 5580

aactactaca ataagatcta ccggaagtct aacaaagaga gcgaggacgg caaggactac 5640

agcatgatca tggcccccac cgtgatcgat tacctgaaca agcggtgcca cggcgagatc 5700

aatggcaact acatctgctg cagctgcaag aatatcggcg cctacaacac cacctccggc 5760

accgtgaaca agaagctgca gaaaaaagaa accgagtgcg aggaagagaa gggccctctg 5820

gacctgatga acgaggtgct gaacaagatg gacaagaagt actccgccca caagatgaag 5880

tgcaccgagg tgtacctgga acacgtggag gaacagctga atgagatcga caacgccatc 5940

aaggattaca agctgtaccc cctggacaga tgcttcgacg accagaccaa gatgaaagtg 6000

tgcgatctga tcgccgacgc catcggctgc aaggataaga caaagctgga tgagctggac 6060

gaatggaacg acatggacct gagaggcacc tacaataagc acaagggcgt gctgatcccc 6120

cccaggcgga gacagctgtg cttcagccgg atcgtgcggg gacccgccaa cctgagaagc 6180

ctgaacgagt tcaaagagga aatcctgaag ggcgctcagt ccgagggcaa gttcctgggc 6240

aattactaca aagagcacaa ggacaaagag aaggccctgg aagccatgaa gaacagcttt 6300

tacgactacg aggacatcat caagggaacc gacatgctga ccaacatcga attcaaggat 6360

atcaagatca agctggaccg gctgctggaa aaagagacaa acaacaccaa gaaagccgag 6420

gactggtgga aaaccaacaa aaagtctatt tggaacgcca tgctgtgcgg ctacaaaaag 6480

agcggcaata agatcattga ccccagctgg tgcaccatcc ctaccaccga gacacccccc 6540

cagtttctga gatggatcaa agaatggggc accaatgtgt gcatccagaa gcaggaacac 6600

aaagaatacg tcaagtctaa gtgctccaac gtgaccaatc tgggagcaca ggccagcgag 6660

agcaacaact gcaccagcga gatcaagaaa taccaggaat ggtcccggaa gcggagcatc 6720

cagtgggaga caatcagcaa gcgctacaag aaatacaagc ggatggacat cctgaaggat 6780

gtgaaagagc ctgacgccaa tacctacctg agagaacact gcagcaagtg cccctgcggc 6840

ttcaacgata tggaagagat gaacaacaac gaggacaatg agaaagaggc cttcaagcag 6900

atcaaagaac aggtgaaaat ccccgccgag ctggaagatg tgatctacag aatcaagcac 6960

cacgagtacg acaagggcaa tgactacatc tgtaacaaat acaagaatat ccacgaccgg 7020

atgaagaaaa acaacggcaa cttcgtgacc gacaatttcg tgaagaagtc ctgggagatt 7080

tccaatggag tgctgatccc tcctcggaga aagaacctgt tcctgtacat cgaccccagc 7140

aagatctgcg aatacaagaa ggaccccaag ctgttcaagg acttcatcta ttggagcgcc 7200

ttcaccgagg tggagcggct gaagaaggcc tacggcggag ccagagccaa ggtggtgcac 7260

gccatgaagt atagcttcac cgacatcggc agcatcatta agggcgacga catgatggaa 7320

aagaatagca gcgacaagat cggcaagatc ctgggggaca ccgacggcca gaacgagaag 7380

cgcaaaaagt ggtgggacat gaacaagtac cacatctggg agagcatgct gtgtggatac 7440

cgggaggccg agggagatac agagactaac gaaaactgcc ggttccccga catcgagagc 7500

gtgcctcagt ttctgcgctg gtttcaggaa tggtcagaga atttttgcga tagaaggcag 7560

aagctgtacg acaagctgaa cagcgagtgc atctccgccg agtgcaccaa tggctccgtg 7620

gacaatagca agtgcaccca cgcctgcgtg aactacaaga attacatcct gaccaaaaag 7680

accgagtacg agatccagac caataagtac gacaacgagt ttaagaacaa gaatagcaac 7740

gataaggacg cccccgacta tctgaaagag aaatgcaacg acaacaagtg cgagtgcctg 7800

aataagcaca ttgacgacaa gaacaaaacc tggaagaacc cctacgagac actggaagat 7860

accttcaaga gcaagtgcga ctgccctaag cccctgccct cccccatcaa gcccgacgat 7920

ctgccacccc aggccgacga gcccttc 7947

<210> 7

<211> 632

<212> PRT

<213> 人工合成序列

<400> 7

Asn Tyr Ile Lys Gly Asp Pro Tyr Phe Ala Glu Tyr Ala Thr Lys Leu

1 5 10 15

Ser Phe Ile Leu Asn Ser Ser Asp Ala Asn Asn Pro Ser Glu Lys Ile

20 25 30

Gln Lys Asn Asn Asp Glu Val Cys Asn Cys Asn Glu Ser Gly Ile Ala

35 40 45

Ser Val Glu Gln Glu Gln Ile Ser Asp Pro Ser Ser Asn Lys Thr Cys

50 55 60

Ile Thr His Ser Ser Ile Lys Ala Asn Lys Lys Lys Val Cys Lys His

65 70 75 80

Val Lys Leu Gly Val Arg Glu Asn Asp Lys Asp Leu Arg Val Cys Val

85 90 95

Ile Glu His Thr Ser Leu Ser Gly Val Glu Asn Cys Cys Cys Gln Asp

100 105 110

Phe Leu Arg Ile Leu Gln Glu Asn Cys Ser Asp Asn Lys Ser Gly Ser

115 120 125

Ser Ser Asn Gly Ser Cys Asn Asn Lys Asn Gln Glu Ala Cys Glu Lys

130 135 140

Asn Leu Glu Lys Val Leu Ala Ser Leu Thr Asn Cys Tyr Lys Cys Asp

145 150 155 160

Lys Cys Lys Ser Glu Gln Ser Lys Lys Asn Asn Lys Asn Trp Ile Trp

165 170 175

Lys Lys Ser Ser Gly Lys Glu Gly Gly Leu Gln Lys Glu Tyr Ala Asn

180 185 190

Thr Ile Gly Leu Pro Pro Arg Thr Gln Ser Leu Cys Leu Val Val Cys

195 200 205

Leu Asp Glu Lys Gly Lys Lys Thr Gln Glu Leu Lys Asn Ile Arg Thr

210 215 220

Asn Ser Glu Leu Leu Lys Glu Trp Ile Ile Ala Ala Phe His Glu Gly

225 230 235 240

Lys Asn Leu Lys Pro Ser His Glu Lys Lys Asn Asp Asp Asn Gly Lys

245 250 255

Lys Leu Cys Lys Ala Leu Glu Tyr Ser Phe Ala Asp Tyr Gly Asp Leu

260 265 270

Ile Lys Gly Thr Ser Ile Trp Asp Asn Glu Tyr Thr Lys Asp Leu Glu

275 280 285

Leu Asn Leu Gln Lys Ile Phe Gly Lys Leu Phe Arg Lys Tyr Ile Lys

290 295 300

Lys Asn Asn Thr Ala Glu Gln Asp Thr Ser Tyr Ser Ser Leu Asp Glu

305 310 315 320

Leu Arg Glu Ser Trp Trp Asn Thr Asn Lys Lys Tyr Ile Trp Leu Ala

325 330 335

Met Lys His Gly Ala Gly Met Asn Ser Thr Thr Cys Cys Gly Asp Gly

340 345 350

Ser Val Thr Gly Ser Gly Ser Ser Cys Asp Asp Ile Pro Thr Ile Asp

355 360 365

Leu Ile Pro Gln Tyr Leu Arg Phe Leu Gln Glu Trp Val Glu His Phe

370 375 380

Cys Lys Gln Arg Gln Glu Lys Val Lys Pro Val Ile Glu Asn Cys Lys

385 390 395 400

Ser Cys Lys Glu Ser Gly Gly Thr Cys Asn Gly Glu Cys Lys Thr Glu

405 410 415

Cys Lys Asn Lys Cys Glu Val Tyr Lys Lys Phe Ile Glu Asp Cys Lys

420 425 430

Gly Gly Asp Gly Thr Ala Gly Ser Ser Trp Val Lys Arg Trp Asp Gln

435 440 445

Ile Tyr Lys Arg Tyr Ser Lys Tyr Ile Glu Asp Ala Lys Arg Asn Arg

450 455 460

Lys Ala Gly Thr Lys Asn Cys Gly Pro Ser Ser Thr Thr Asn Ala Ala

465 470 475 480

Glu Asn Lys Cys Val Gln Ser Asp Ile Asp Ser Phe Phe Lys His Leu

485 490 495

Ile Asp Ile Gly Leu Thr Thr Pro Ser Ser Tyr Leu Ser Ile Val Leu

500 505 510

Asp Asp Asn Ile Cys Gly Ala Asp Lys Ala Pro Trp Thr Thr Tyr Thr

515 520 525

Thr Tyr Thr Thr Thr Glu Lys Cys Asn Lys Glu Thr Asp Lys Ser Lys

530 535 540

Leu Gln Gln Cys Asn Thr Ala Val Val Val Asn Val Pro Ser Pro Leu

545 550 555 560

Gly Asn Thr Pro His Gly Tyr Lys Tyr Ala Cys Gln Cys Lys Ile Pro

565 570 575

Thr Asn Glu Glu Thr Cys Asp Asp Arg Lys Glu Tyr Met Asn Gln Trp

580 585 590

Ser Cys Gly Ser Ala Arg Thr Met Lys Arg Gly Tyr Lys Asn Asp Asn

595 600 605

Tyr Glu Leu Cys Lys Tyr Asn Gly Val Asp Val Lys Pro Thr Thr Val

610 615 620

Arg Ser Asn Ser Ser Lys Leu Asp

625 630

<210> 8

<211> 1896

<212> DNA

<213> 人工合成序列

<400> 8