本發(fā)明屬于生物產(chǎn)品制備技術(shù)領(lǐng)域，涉及一種牛呼吸道胞合體病毒抗原蛋白及其制備方法。

背景技術(shù)：

牛呼吸道胞合體病毒（Bovine Respiration Syncytial Virus – BRSV）感染可引起牛呼吸道疾病，在歐共體國(guó)家被列為僅次于牛粘膜?。˙VD）及牛傳染性鼻氣管炎（IBR）的重要牛病之一。本病呈世界范圍性流行，其危害性在于BRSV感染發(fā)病率高達(dá)到60%-80%，死亡率在一些爆發(fā)中達(dá)到20%以上，給養(yǎng)牛業(yè)造成很大的經(jīng)濟(jì)損失。雖然這種BRSV從上個(gè)世紀(jì)70年代就開(kāi)始列入研發(fā)范疇，由于種種原因并沒(méi)有開(kāi)發(fā)出理想的藥物和疫苗。盡管已有關(guān)于BRSV的多種滅活，弱毒，重組以及DNA疫苗的報(bào)道，但距離臨床使用還有一定距離，目前國(guó)際上尚沒(méi)有理想的病毒疫苗。

傳統(tǒng)疫苗的研制和生產(chǎn)主要是通過(guò)改變培養(yǎng)條件，或在不同寄主動(dòng)物上傳代使致病微生物毒性減弱，或通過(guò)物理、化學(xué)方法將其滅活來(lái)完成的。隨著人類(lèi)知識(shí)的不斷進(jìn)步，傳統(tǒng)疫苗的局限性也日益顯露出來(lái)：(1)動(dòng)物和人類(lèi)的病毒需要在動(dòng)物細(xì)胞中培養(yǎng)，這使得疫苗生產(chǎn)的成本很高；(2)疫苗中的致病物質(zhì)在疫苗生產(chǎn)過(guò)程中有可能沒(méi)有完全殺死或充分減毒，這會(huì)導(dǎo)致疫苗中含有強(qiáng)毒性致病物質(zhì)，進(jìn)而使得疾病在更大的范圍內(nèi)傳播；(3)減毒菌株有可能會(huì)發(fā)生突變，引起病害。

牛呼吸道胞合體病毒是副黏液病毒科家族的成員，其上的F-蛋白是高度保守的并形成三聚體刺突，其在激活下發(fā)生構(gòu)象變化。F-蛋白介導(dǎo)病毒和細(xì)胞膜的融合，讓病毒核殼體進(jìn)入細(xì)胞質(zhì)，抑制F-蛋白介導(dǎo)的步驟阻抑感染周期的起始階段并中和病毒感染性可以有效的防止病毒侵害，即針對(duì)F-蛋白的抗體，抑制它的活性，可以中和病毒感染性并可以保護(hù)免受BRSV 感染。施一公開(kāi)創(chuàng)了中國(guó)結(jié)構(gòu)生物學(xué)基礎(chǔ)理論研究的先河，本發(fā)明將結(jié)構(gòu)生物學(xué)應(yīng)用到實(shí)際中，合成牛呼吸道胞合體病毒抗原蛋白。

技術(shù)實(shí)現(xiàn)要素：

本發(fā)明的目的在于克服現(xiàn)有技術(shù)存在的缺點(diǎn)，提供一種牛呼吸道胞合體病毒抗原蛋白及其制備方法，該抗原蛋白應(yīng)用蛋白質(zhì)晶體學(xué)的技術(shù)和理論，確定蛋白在病毒侵染和致病過(guò)程中的結(jié)構(gòu)變化，再根據(jù)結(jié)構(gòu)變化的結(jié)果對(duì)這個(gè)關(guān)鍵的病毒蛋白進(jìn)行基因工程改造，使其成為只侵染不致病、并引起動(dòng)物體抗體高效反應(yīng)的蛋白抗原。

為了實(shí)現(xiàn)上述目的，本發(fā)明解決其技術(shù)問(wèn)題所采取的技術(shù)方案是：

一種牛呼吸道胞合體病毒抗原蛋白，編碼牛呼吸道胞合體病毒關(guān)鍵蛋白F-蛋白野生種的氨基酸序列簡(jiǎn)稱SEQ AAA42804.1，具體序列為MATTTMRMIISIILISTYVPHITLCQNITEEFYQSTCSAVSRGYLSALRTGWYTSVVTIELSKIQKNVCNGTDSKVKLIKQELERYNNAVAELQSLMQNEPTSSSRAKRGIPESIHYTRNSTKKFYGLMGKKRKRRFLGFLLGIGSAIASGVAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKNYIDKELLPKVNNHDCRISNIATVIEFQQKNNRLLEIAREFSVNAGITTPLSTYMLTNSELLSIINDMPITNDQKKLMSVCQIVRQQSYSIMSVLREVIAYVVQLPLYGVIDTPCWKLHTSPLCTTDNKEGSNICLTRTDRGWYCDNAGSVSFFPQAETCKVQSNRVFCDTMNSLTLPTDVNLCNTDIFNSKYDCKIMTSKTDISSSVITSIGAIVSCYGKTKCTASNKNRGIIKTFSNGCDYVSNKGVDTVSVGNTLYYVNKLEGKALYIKGEPIINYYNPLVFPSDEFDASIAQVNAKINQSLAFIRRSDELLHSVDVGKSTTNVVITTIIIVIVVVILMLITVGLLFYCKTRSTPIMLGKDQLSSINNLSFSK，其特征在于：所述牛呼吸道胞合體病毒抗原蛋白的氨基酸序列相比SEQ AAA42804.1至少有兩個(gè)位點(diǎn)被半胱氨酸替代，且替代后的半胱氨酸之間由二硫鍵連接。

作為優(yōu)化的，所述位點(diǎn)為氨基酸序列第148位纈氨酸和288位異亮氨酸。

作為優(yōu)化的，所述位點(diǎn)為氨基酸序列第158位亮氨酸和290位絲氨酸。

作為優(yōu)化的，所述牛呼吸道胞合體病毒抗原蛋白的氨基酸序列相比SEQ AAA42804.1，在第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充。

作為優(yōu)化的，所述針對(duì)牛呼吸道胞合體病毒抗原蛋白的氨基酸序列剔除SEQ AAA42804.1序列中的103位點(diǎn)至138位點(diǎn)的氨基酸序列，采用sgsgs連接102位點(diǎn)和139位點(diǎn)。

作為優(yōu)化的，所述針對(duì)牛呼吸道胞合體病毒抗原蛋白的氨基酸序列剔除SEQ AAA42804.1序列中的103位點(diǎn)至142位點(diǎn)的氨基酸序列，采用sgsgs連接102位點(diǎn)和143位點(diǎn)。

作為優(yōu)化的，所述編碼牛呼吸道胞合體病毒抗原蛋白的氨基酸序列相比SEQ AAA42804.1序列，第99位點(diǎn)天冬氨酸由半胱氨酸替代，第362位點(diǎn)絲氨酸由半胱氨酸替代，并在替代后的半胱氨酸之間連接內(nèi)二硫鍵。

一種牛呼吸道胞合體病毒抗原蛋白的制備方法，其特征在于：具體工藝如下：

（1）根據(jù)結(jié)構(gòu)生物學(xué)的原理，進(jìn)行蛋白結(jié)晶，結(jié)晶后用X-射線觀測(cè)，確定侵染病毒關(guān)鍵病原F-蛋白侵染前后的結(jié)構(gòu)變化；

（2）根據(jù)步驟（1）結(jié)構(gòu)變化設(shè)計(jì)出抗原蛋白的序列和結(jié)構(gòu)；

（3）經(jīng)多種抗原測(cè)試和穩(wěn)定性特性檢測(cè)確定出需要抗原蛋白的合格性；

（4）將經(jīng)過(guò)驗(yàn)證的抗原蛋白序列進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純和檢測(cè)，得到用來(lái)作為動(dòng)物試驗(yàn)和臨床試驗(yàn)的針對(duì)牛呼吸道胞合體病毒抗原蛋白。

牛胞合體病毒關(guān)鍵F-蛋白介導(dǎo)病毒和細(xì)胞膜的融合，讓病毒核殼體進(jìn)入細(xì)胞。針對(duì)牛呼吸道胞合體病毒設(shè)計(jì)的抗原蛋白，便是根據(jù)F-蛋白侵染前后的結(jié)構(gòu)變化設(shè)定的，在基因工程的基礎(chǔ)上使F-蛋白結(jié)構(gòu)始終保持在融合前的狀態(tài)，使其具有只侵染不致病、并引起動(dòng)物體抗體高效反應(yīng)的蛋白抗原等特點(diǎn)。

本發(fā)明能夠使F-蛋白在侵染前后都保持三聚體刺突狀態(tài)（閉合狀態(tài)），得到只侵染不致病、并引起動(dòng)物抗體高效反應(yīng)的蛋白抗原；為了保證來(lái)自于病原病毒上的F-蛋白處在穩(wěn)定的侵染前的狀態(tài)，給F-蛋白上增添了系列的連接鏈，包括內(nèi)二硫鍵、二硫鍵、空穴填充和單鏈連接。

本發(fā)明與現(xiàn)有技術(shù)相比，其有益效果是：采用蛋白質(zhì)晶體學(xué)方法制備的牛呼吸道胞合體病毒抗原蛋白能夠有效的預(yù)防和治療牛呼吸道胞合體病毒感染，以此為基礎(chǔ)可以開(kāi)發(fā)出理想的病毒疫苗；有效控制和防范病毒感染帶來(lái)的損失，具有重大的經(jīng)濟(jì)意義和社會(huì)意義。

附圖說(shuō)明

圖1 本發(fā)明涉及利用Phenix軟件進(jìn)行牛合胞體病毒F-蛋白的三維立體結(jié)構(gòu)模擬圖，左圖為浸染前三聚體三維立體結(jié)構(gòu)模擬圖，右圖為左圖右下角方框位置的局部放大圖。

圖2 本發(fā)明涉及的牛合胞體病毒呼吸道病毒侵染前后過(guò)程的關(guān)鍵的F-蛋白結(jié)構(gòu)變化示意圖。在圖中顯示侵染前（左），F(xiàn)-蛋白三聚體呈現(xiàn)三聚體刺突狀態(tài)，侵染后（右）F-蛋白被激活，發(fā)生構(gòu)象變化，病毒和細(xì)胞膜的融合，病毒核殼體進(jìn)入細(xì)胞質(zhì)，進(jìn)而侵染牛體并使其致病。

圖3 本發(fā)明涉及的經(jīng)過(guò)改造成功的牛呼吸道胞合體蛋白抗原侵染牛體后的電鏡照片狀態(tài)。左圖顯示RD-BRSV-DB1-CF1改造的牛呼吸道胞合體蛋白抗原侵染牛體后的狀態(tài)，可見(jiàn)呈現(xiàn)穩(wěn)定的融合前三聚體刺突狀態(tài)，右圖為未經(jīng)過(guò)改造的牛呼吸道胞合體病毒侵染牛體后的電鏡照片。

圖4 本發(fā)明涉及的F-蛋白上添加連接鏈?zhǔn)疽鈭D，包括內(nèi)二硫鍵、二硫鍵、空穴填充和單鏈連接，這些鏈接保證F-蛋白保持三聚體刺突狀態(tài)。

具體實(shí)施方式

下面通過(guò)實(shí)施例并結(jié)合附圖對(duì)本發(fā)明進(jìn)一步說(shuō)明。

實(shí)施例1：將SEQ AAA42804.1進(jìn)行如下改造得到本發(fā)明的一種牛呼吸道胞合體病毒抗原蛋白：

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第148位纈氨酸由半胱氨酸替代，第288位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接，將其進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB1，其氨基酸序列簡(jiǎn)稱為SEQ DB1。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第158位亮氨酸由半胱氨酸替代，第290位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接，將其進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB2，其氨基酸序列簡(jiǎn)稱為SEQ DB2。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第148位纈氨酸由半胱氨酸替代，第288位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接，同時(shí)將第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充，將其進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB1-CF1，其氨基酸序列簡(jiǎn)稱為SEQ DB1-CF1。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第158位亮氨酸由半胱氨酸替代，第290位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接，同時(shí)將第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充，將其進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB2-CF1，其氨基酸序列簡(jiǎn)稱為SEQ DB2-CF1。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第148位纈氨酸由半胱氨酸替代，第288位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接；同時(shí)將第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充；剔除SEQ AAA42804.1序列中103至位點(diǎn)138位點(diǎn)的氨基酸序列，采用sgsgs連接102及139位點(diǎn)，將其進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB1-CF1-SC1，其氨基酸序列簡(jiǎn)稱為SEQ DB1-CF1-SC1。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第158位亮氨酸由半胱氨酸替代，第290位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接，同時(shí)將第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充；剔除SEQ AAA42804.1序列中103至位點(diǎn)138位點(diǎn)的氨基酸序列，采用sgsgs連接102及139位點(diǎn)，將其進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB2-CF1-SC1，其氨基酸序列簡(jiǎn)稱為SEQ DB2-CF1-SC1。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第148位纈氨酸由半胱氨酸替代，第288位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接；同時(shí)將第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充；剔除SEQ AAA42804.1序列中103至位點(diǎn)138位點(diǎn)的氨基酸序列，采用sgsgs連接102及139位點(diǎn)，第99位點(diǎn)天冬氨酸由半胱氨酸替代，第362位點(diǎn)絲氨酸由半胱氨酸替代，并在替代后的半胱氨酸之間連接內(nèi)二硫鍵，進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB1-CF1-SC1-ID1，其氨基酸序列簡(jiǎn)稱為SEQ DB1-CF1-SC1-ID1。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第148位纈氨酸由半胱氨酸替代，第288位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接；同時(shí)將第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充；剔除SEQ AAA42804.1序列中103至位點(diǎn)142位點(diǎn)的氨基酸序列，采用sgsgs連接102及143位點(diǎn)，第99位點(diǎn)天冬氨酸由半胱氨酸替代，第362位點(diǎn)絲氨酸由半胱氨酸替代，并在替代后的半胱氨酸之間連接內(nèi)二硫鍵，進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB1-CF1-SC2-ID1，其氨基酸序列簡(jiǎn)稱為SEQ DB1-CF1-SC2-ID1。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第158位亮氨酸由半胱氨酸替代，第290位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接，同時(shí)將第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充；剔除SEQ AAA42804.1序列中103至位點(diǎn)138位點(diǎn)的氨基酸序列，采用sgsgs連接102及139位點(diǎn)；第99位點(diǎn)天冬氨酸由半胱氨酸替代，第362位點(diǎn)絲氨酸由半胱氨酸替代，并在替代后的半胱氨酸之間連接內(nèi)二硫鍵。其進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB2-CF1-SC1-ID1，其氨基酸序列簡(jiǎn)稱為SEQ DB2-CF1-SC1-ID1。

牛呼吸道胞合體病毒F-蛋白氨基酸序列SEQ AAA42804.1第158位亮氨酸由半胱氨酸替代，第290位異亮氨酸由半胱氨酸替代，并由二硫鍵將兩位點(diǎn)連接，同時(shí)將第260位點(diǎn)的亮氨酸由半胱氨酸替代，并在該位點(diǎn)實(shí)現(xiàn)空穴填充；剔除SEQ AAA42804.1序列中103至位點(diǎn)142位點(diǎn)的氨基酸序列，采用sgsgs連接102及143位點(diǎn)；第99位點(diǎn)天冬氨酸由半胱氨酸替代，第362位點(diǎn)絲氨酸由半胱氨酸替代，并在替代后的半胱氨酸之間連接內(nèi)二硫鍵。其進(jìn)行相應(yīng)的蛋白合成、表達(dá)、提純，得到的牛呼吸道胞合體病毒抗原蛋白簡(jiǎn)稱RD-BRSV-DB2-CF1-SC2-ID1，其氨基酸序列簡(jiǎn)稱為SEQ DB2-CF1-SC2-ID1。

免疫原性又稱抗原性，指抗原刺激機(jī)體產(chǎn)生免疫應(yīng)答能力的特性。免疫反應(yīng)性指抗原分子能與相應(yīng)免疫應(yīng)答的產(chǎn)物（抗體或致敏淋巴細(xì)胞），在體內(nèi)或體外發(fā)生特異性結(jié)合的性能，采用ELISA法對(duì)抗原與抗體的親和力-免疫進(jìn)行檢驗(yàn)。ELISA法具有靈敏、特異、簡(jiǎn)單、快速、穩(wěn)定及易于自動(dòng)化操作等特點(diǎn)，作為免疫診斷中的一項(xiàng)新技術(shù)，已成功地應(yīng)用于大分子抗原和小分子抗原的定量測(cè)定。高親合力的抗體與抗原的結(jié)合力強(qiáng)，即抗原濃度很低時(shí)也有較多的抗體結(jié)合抗原形成免疫復(fù)合物。親和性用平衡常數(shù)K來(lái)表示，K值越大，親和性越高，與抗原結(jié)合也越牢固。表1給出了本發(fā)明制備的抗原蛋白分別在Site?、SiteQP、SiteⅤ、SiteⅡ位點(diǎn)與抗體結(jié)合的親和性數(shù)據(jù)。

表1 實(shí)施例牛呼吸道胞合體病毒抗原蛋白的特性檢測(cè)

本發(fā)明經(jīng)過(guò)設(shè)計(jì)改造的F-蛋白具有較高的抗體庫(kù)親和性。經(jīng)過(guò)改造后的抗原蛋白都保持融合前三聚體刺突狀態(tài)（Trimer），且都能得到目的抗原蛋白。

本發(fā)明獲得純化的抗原蛋白與D25抗體進(jìn)行特異性結(jié)合，經(jīng)蛋白免疫電泳和ELISA方法檢測(cè)，結(jié)果表明不僅具有較好的免疫原性，而且能夠誘導(dǎo)小鼠產(chǎn)生較高的抗體水平，表2給出了D25抗體與本發(fā)明抗原蛋白特異性結(jié)合的免疫原性加權(quán)平均值，對(duì)照組為非變異野生種。

表2 實(shí)施例牛呼吸道胞合體病毒抗原蛋白與D25特異性結(jié)合的特性檢測(cè)

對(duì)本發(fā)明中的抗原蛋白進(jìn)行物理化學(xué)穩(wěn)定性進(jìn)行測(cè)定，包括溫度、pH、滲透壓等方面，詳細(xì)數(shù)據(jù)見(jiàn)表3，表中數(shù)據(jù)為在相應(yīng)條件下的抗原蛋白活性與最佳抗原蛋白活性的比值，結(jié)果表明經(jīng)過(guò)設(shè)計(jì)改造的抗原蛋白對(duì)溫度、PH值、滲透壓等呈現(xiàn)較好的穩(wěn)定性，具有較高的反應(yīng)活性。

表3實(shí)施例牛呼吸道胞合體病毒抗原蛋白的反應(yīng)活性穩(wěn)定性

實(shí)施例2：制備實(shí)施例1中RD-BRSV-DB1-CF1-SC1-ID1的具體工藝?？吹降鞍讬C(jī)構(gòu)和形狀可以通過(guò)以下三種途徑，分別為x-射線（x-ray Crystallography）、核磁共振（NMR）、電鏡，本發(fā)明采用X-射線法對(duì)蛋白結(jié)構(gòu)進(jìn)行觀察。采用Solution maker 600 plux系列將目標(biāo)蛋白進(jìn)行結(jié)晶，將結(jié)晶后的目標(biāo)蛋白采用X-射線系統(tǒng)（SER-CAT22）收集結(jié)晶體數(shù)據(jù)，并使其成像，獲得蛋白結(jié)構(gòu)的電子云圖，得到關(guān)鍵蛋白F-蛋白侵染前后的數(shù)據(jù)變化，侵染前后結(jié)構(gòu)變化如圖 2所示。

對(duì)上述電子云圖進(jìn)行云數(shù)據(jù)處理，利用相應(yīng)的軟件（Phenix）和野生蛋白序列進(jìn)行數(shù)據(jù)模擬，形成蛋白結(jié)構(gòu)的三維立體結(jié)構(gòu)模擬圖形。根據(jù)蛋白序列模擬出的三維蛋白結(jié)構(gòu)圖形得到F-蛋白的三聚體結(jié)構(gòu)，詳見(jiàn)圖1。

按照晶體化學(xué)的原理，利用模擬軟件進(jìn)行抗原蛋白結(jié)構(gòu)設(shè)計(jì)，其設(shè)計(jì)包括如圖4所示的四種方式：內(nèi)二硫鍵（intra disulphide bond）、二硫鍵（disulphide bond）、空穴填充（cavity filling）和單鏈連接（single chain），對(duì)野生種AAA42804.1進(jìn)行設(shè)計(jì)。

將設(shè)計(jì)好的蛋白序列轉(zhuǎn)譯成DNA序列，采用invitrogen公司提供的Expi293F作為細(xì)胞載體在293Fectin培養(yǎng)基中進(jìn)行培養(yǎng)5天，收集細(xì)胞培養(yǎng)液，將目標(biāo)蛋白進(jìn)行提純精制得到設(shè)計(jì)好的純化的BRSV F-蛋白，即RD-BRSV-DB1-CF1-SC1-ID1，改造后的牛呼吸道胞合體蛋白抗原侵染牛體后呈現(xiàn)穩(wěn)定的融合前三聚體刺突狀態(tài)見(jiàn)圖3（左）。

上述具體實(shí)施方式僅是本發(fā)明的具體個(gè)案，本發(fā)明的專(zhuān)利保護(hù)范圍包括但不限于上述具體實(shí)施方式的產(chǎn)品形態(tài)和式樣，任何符合本發(fā)明權(quán)利要求書(shū)的一種牛呼吸道胞合體病毒抗原蛋白且任何所屬技術(shù)領(lǐng)域的普通技術(shù)人員對(duì)其所做的適當(dāng)變化或修飾，皆應(yīng)落入本發(fā)明的專(zhuān)利保護(hù)范圍。

SEQUENCE LISTING

<110> 煙臺(tái)偌帝生物工程有限公司

<120> 一種牛呼吸道胞合體病毒抗原蛋白

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Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Val Cys Gln Ile Val Arg

245 250 255

Gln Gln Ser Tyr Ser Cys Met Ser Val Leu Arg Glu Val Ile Ala Tyr

260 265 270

Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys

275 280 285

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

290 295 300

Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly

305 310 315 320

Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn

325 330 335

Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val

340 345 350

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

355 360 365

Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly

370 375 380

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

385 390 395 400

Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser

405 410 415

Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val

420 425 430

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

435 440 445

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

450 455 460

Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg

465 470 475 480

Arg Ser Asp Glu Leu Leu His

485

<210> 5

<211> 487

<212> PRT

<213> 人工序列

<400> 5

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

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

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ala Glu Leu Gln Ser Leu Met Gln Asn Glu Pro Thr Ser Ser Ser

65 70 75 80

Arg Ala Lys Arg Gly Ile Pro Glu Ser Ile His Tyr Thr Arg Asn Ser

85 90 95

Thr Lys Lys Phe Tyr Gly Leu Met Gly Lys Lys Arg Lys Arg Arg Phe

100 105 110

Leu Gly Phe Leu Leu Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala

115 120 125

Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn

130 135 140

Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val

145 150 155 160

Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys

165 170 175

Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile

180 185 190

Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile

195 200 205

Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr

210 215 220

Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro

225 230 235 240

Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Val Cys Gln Ile Val Arg

245 250 255

Gln Gln Ser Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr

260 265 270

Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys

275 280 285

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

290 295 300

Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly

305 310 315 320

Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn

325 330 335

Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val

340 345 350

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

355 360 365

Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile Gly

370 375 380

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

385 390 395 400

Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser

405 410 415

Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val

420 425 430

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

435 440 445

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

450 455 460

Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg

465 470 475 480

Arg Ser Asp Glu Leu Leu His

485

<210> 6

<211> 456

<212> PRT

<213> 人工序列

<400> 6

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

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

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

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

65 70 75 80

Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val

85 90 95

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

100 105 110

Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

115 120 125

Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

130 135 140

Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

Ile Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

165 170 175

Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser

180 185 190

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met

195 200 205

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Val Cys Gln Ile Val

210 215 220

Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Leu Arg Glu Val Ile Ala

225 230 235 240

Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp

245 250 255

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

260 265 270

Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala

275 280 285

Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser

290 295 300

Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp

305 310 315 320

Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys

325 330 335

Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile

340 345 350

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

355 360 365

Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val

370 375 380

Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr

385 390 395 400

Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile

405 410 415

Ile Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala

420 425 430

Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile

435 440 445

Arg Arg Ser Asp Glu Leu Leu His

450 455

<210> 7

<211> 452

<212> PRT

<213> 人工序列

<400> 7

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

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

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

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

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu

130 135 140

Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Ala Thr Val

145 150 155 160

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

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

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

210 215 220

Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr Val Val Gln

225 230 235 240

Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr

245 250 255

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

260 265 270

Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser

275 280 285

Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe

290 295 300

Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys

305 310 315 320

Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu

385 390 395 400

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

405 410 415

Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln

420 425 430

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

435 440 445

Glu Leu Leu His

450

<210> 8

<211> 456

<212> PRT

<213> 人工序列

<400> 8

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

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

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ala Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Thr Ser Gly Ser

65 70 75 80

Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val

85 90 95

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

100 105 110

Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

115 120 125

Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

130 135 140

Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

Ile Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

165 170 175

Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser

180 185 190

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met

195 200 205

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Val Cys Gln Ile Val

210 215 220

Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Leu Arg Glu Val Ile Ala

225 230 235 240

Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp

245 250 255

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

260 265 270

Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala

275 280 285

Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys

290 295 300

Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp

305 310 315 320

Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys

325 330 335

Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile

340 345 350

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

355 360 365

Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val

370 375 380

Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr

385 390 395 400

Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile

405 410 415

Ile Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala

420 425 430

Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile

435 440 445

Arg Arg Ser Asp Glu Leu Leu His

450 455

<210> 9

<211> 452

<212> PRT

<213> 人工序列

<400> 9

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

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

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ala Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Thr Ser Gly Ser

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu

130 135 140

Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Ala Thr Val

145 150 155 160

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

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

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

210 215 220

Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr Val Val Gln

225 230 235 240

Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr

245 250 255

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

260 265 270

Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser

275 280 285

Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys Asn Arg Val Phe

290 295 300

Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys

305 310 315 320

Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu

385 390 395 400

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

405 410 415

Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln

420 425 430

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

435 440 445

Glu Leu Leu His

450

<210> 10

<211> 456

<212> PRT

<213> 人工序列

<400> 10

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

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

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ala Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Thr Ser Gly Ser

65 70 75 80

Gly Ser Gly Phe Leu Leu Gly Ile Gly Ser Ala Cys Ala Ser Gly Val

85 90 95

Ala Val Ser Lys Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys

100 105 110

Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly

115 120 125

Val Ser Val Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp

130 135 140

Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn

145 150 155 160

Ile Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu

165 170 175

Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser

180 185 190

Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met

195 200 205

Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Val Cys Gln Ile Val

210 215 220

Arg Gln Gln Ser Tyr Ser Cys Met Ser Val Leu Arg Glu Val Ile Ala

225 230 235 240

Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp

245 250 255

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

260 265 270

Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala

275 280 285

Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys

290 295 300

Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp

305 310 315 320

Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys

325 330 335

Ile Met Thr Ser Lys Thr Asp Ile Ser Ser Ser Val Ile Thr Ser Ile

340 345 350

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

355 360 365

Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val

370 375 380

Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr

385 390 395 400

Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile

405 410 415

Ile Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala

420 425 430

Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile

435 440 445

Arg Arg Ser Asp Glu Leu Leu His

450 455

<210> 11

<211> 452

<212> PRT

<213> 人工序列

<400> 11

Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys Ser Ala Val Ser

1 5 10 15

Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Val

20 25 30

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

35 40 45

Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg Tyr Asn Asn Ala

50 55 60

Val Ala Glu Leu Gln Ser Leu Met Gln Cys Glu Pro Thr Ser Gly Ser

65 70 75 80

Gly Ser Gly Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys

85 90 95

Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu

100 105 110

Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu

115 120 125

Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu

130 135 140

Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Ala Thr Val

145 150 155 160

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

165 170 175

Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu

180 185 190

Thr Asn Ser Glu Leu Leu Ser Phe Ile Asn Asp Met Pro Ile Thr Asn

195 200 205

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

210 215 220

Tyr Ser Ile Met Cys Val Leu Arg Glu Val Ile Ala Tyr Val Val Gln

225 230 235 240

Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr

245 250 255

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

260 265 270

Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser

275 280 285

Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Cys Asn Arg Val Phe

290 295 300

Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys

305 310 315 320

Asn Thr Asp Ile Phe Asn Ser Lys Tyr Asp Cys Lys Ile Met Thr Ser

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu

385 390 395 400

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

405 410 415

Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln

420 425 430

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

435 440 445

Glu Leu Leu His

450