本發(fā)明涉及病毒疫苗生產(chǎn)技術(shù)領(lǐng)域，具體涉及一種口蹄疫病毒嵌合病毒樣顆粒及其制備方法。

背景技術(shù)：

病毒樣顆粒（virus-likeparticles，vlps）是一種含有某種病毒一種或多種結(jié)構(gòu)蛋白的空心顆?；虬铑w粒結(jié)構(gòu)。vlps不含病毒核酸，且不能自主復(fù)制，不存在基因重組或重配和毒力回復(fù)的可能，作為疫苗使用時(shí)具有較高的安全性。同時(shí)，vlps在形態(tài)結(jié)構(gòu)上與天然病毒顆粒相同或相似，可通過(guò)和天然病毒感染相同的途徑呈遞給免疫細(xì)胞，有效地誘導(dǎo)機(jī)體的免疫系統(tǒng)產(chǎn)生免疫保護(hù)反應(yīng)。目前，國(guó)內(nèi)外相關(guān)研究已利用昆蟲(chóng)桿狀病毒表達(dá)系統(tǒng)在體外成功表達(dá)并制備了o型口蹄疫病毒的vlps，但研究同時(shí)顯示，相比于其它血清型的口蹄疫病毒，o型口蹄疫病毒vlps的耐酸性較低，在ph值較低的昆蟲(chóng)細(xì)胞中表達(dá)時(shí)其表達(dá)量會(huì)受到影響，而且表達(dá)后vlps的組裝效率較差。

技術(shù)實(shí)現(xiàn)要素：

本發(fā)明的目的就是針對(duì)上述現(xiàn)有技術(shù)中的缺陷，提供了一種口蹄疫病毒嵌合病毒樣顆粒及其制備方法。

為了實(shí)現(xiàn)上述目的，本發(fā)明提供的技術(shù)方案為：一種口蹄疫病毒嵌合病毒樣顆粒，所述嵌合病毒樣顆粒為口蹄疫病毒嵌合蛋白，所述嵌合蛋白的氨基酸序列如序列表中seqidno.1所示。

本發(fā)明的第二個(gè)目的是提供了上述一種口蹄疫病毒嵌合病毒樣顆粒的制備方法，包括以下步驟：

1）合成嵌合衣殼蛋白p12a基因：

以a型口蹄疫病毒f72株的衣殼蛋白基因p12a為骨架載體，將其結(jié)構(gòu)蛋白vp1基因替換為o型口蹄疫病毒by/cha/2010株的vp1基因，從而形成o-a嵌合的口蹄疫病毒衣殼蛋白序列p12a(o-vp1)，其dna序列如序列表中seqidno.2所示；

2）合成3c基因：

3c蛋白酶的氨基酸序列如序列表中seqidno.3所示，其dna序列如序列表中seqidno.4所示；

3）重組轉(zhuǎn)移載體pfastbacdual-p12a3c(o-vp1)的構(gòu)建：

將步驟1）得到的o-a嵌合的口蹄疫病毒衣殼蛋白p12a(o-vp1)與桿狀病毒表達(dá)載體pfastbacdual同時(shí)用限制性?xún)?nèi)切酶ecoriandhindiii雙酶切，回收后的p12a(o-vp1)片段和pfastbacdual片段連接后轉(zhuǎn)化top10感受態(tài)細(xì)胞，挑取單克隆菌落后搖菌提取質(zhì)粒，將雙酶切及測(cè)序鑒定為陽(yáng)性的重組質(zhì)粒命名為pfastbacdual-p12a(o-vp1)；然后將重組質(zhì)粒pfastbacdual-p12a(o-vp1)和步驟2）得到的3c基因片段分別用限制性?xún)?nèi)切酶ncoi和kpni雙酶切，回收酶切產(chǎn)物并連接，最后將鑒定正確的重組轉(zhuǎn)移載體命名為pfastbacdual-p12a3c(o-vp1)；

4）重組桿狀病毒rbacmid-p12a3c(o-vp1)的構(gòu)建：

將步驟3）得到的重組轉(zhuǎn)移載體pfastbacdual-p12a3c(o-vp1)轉(zhuǎn)化dh10bac?escherichiacoli感受態(tài)細(xì)胞后進(jìn)行藍(lán)白斑篩選，挑選陽(yáng)性克隆并提取重組桿狀病毒質(zhì)粒rbacmid-p12a3c(o-vp1)，利用通用引物m13f/r進(jìn)行pcr鑒定；

5）重組桿狀病毒rbac-p12a3c(o-vp1)的構(gòu)建：

步驟4）得到的重組桿狀病毒質(zhì)粒rbacmid-p12a3c(o-vp1)在cellfectin?iireagent轉(zhuǎn)染試劑的協(xié)助下轉(zhuǎn)染sf9細(xì)胞，置于27℃細(xì)胞培養(yǎng)箱中培養(yǎng)72小時(shí)，sf9細(xì)胞出現(xiàn)病變后，將細(xì)胞吹打下來(lái)置于離心管中劇烈振蕩，除去細(xì)胞和碎片并收集上清液，即得到第一代重組桿狀病毒rbac-dual-p12a3c和rbac-dual，將重組桿狀病毒在sf9細(xì)胞中傳代培養(yǎng)至第三代，保存?zhèn)溆谩?/p>

本發(fā)明的有益效果為：本發(fā)明首次以耐酸性較高的a型口蹄疫病毒衣殼蛋白為載體骨架，將o型口蹄疫病毒主要抗原蛋白vp1嵌合至a型口蹄疫病毒衣殼蛋白中，并在昆蟲(chóng)桿狀病毒表達(dá)系統(tǒng)中成功的表達(dá)了該嵌合vlps，進(jìn)一步將其做為疫苗免疫豚鼠，使豚鼠獲得了較高的抗o型口蹄疫病毒的體液和細(xì)胞免疫水平，并在攻毒實(shí)驗(yàn)中產(chǎn)生了較高的免疫保護(hù)率，為今后口蹄疫新型疫苗的開(kāi)發(fā)提供了新的思路和方法。

附圖說(shuō)明

圖1顯示為重組桿粒及重組桿狀病毒的pcr鑒定結(jié)果。

其中，圖a中，lanem:dnamarker；lanep12a3c:通用引物m13-f/r擴(kuò)增重組桿粒；圖b中，lane3c:特異性引物3c-f/r擴(kuò)增重組桿狀病毒；lanep12a:特異性引物p12a-f/r擴(kuò)增重組桿狀病毒。

圖2顯示為重組桿狀病毒感染sf9細(xì)胞后第0、12、24及36小時(shí)后重組蛋白表達(dá)情況的免疫熒光檢查結(jié)果。

圖3顯示為重組蛋白的western-blot檢測(cè)結(jié)果。

圖4顯示為嵌合vlps的電鏡觀察結(jié)果。

圖5顯示為嵌合vlps疫苗免疫豚鼠后第0,7,14,21及28天的血清igg抗體水平間接elisa檢測(cè)結(jié)果。

圖6顯示為嵌合vlps疫苗免疫豚鼠后第28天的血清中和抗體水平檢測(cè)結(jié)果。

圖7顯示為嵌合vlps疫苗免疫豚鼠后第28天的血清細(xì)胞因子水平檢測(cè)結(jié)果。

圖8顯示為嵌合vlps疫苗免疫豚鼠后第28天的t淋巴細(xì)胞增殖水平檢測(cè)結(jié)果。

具體實(shí)施方式

實(shí)施例1：

一種口蹄疫病毒嵌合病毒樣顆粒，其為口蹄疫病毒嵌合蛋白，嵌合蛋白的氨基酸序列如序列表中seqidno.1所示，口蹄疫病毒嵌合病毒樣顆粒的制備方法，包括以下步驟：

1、嵌合衣殼蛋白p12a-3c基因的設(shè)計(jì)及合成：

以a型口蹄疫病毒f72株的衣殼蛋白基因p12a為骨架載體，將其結(jié)構(gòu)蛋白vp1基因替換為o型口蹄疫病毒by/cha/2010株的vp1基因，從而形成o-a嵌合的口蹄疫病毒衣殼蛋白序列p12a(o-vp1)。為了在昆蟲(chóng)細(xì)胞中準(zhǔn)確并高效的表達(dá)嵌合衣殼蛋白序列p12a(o-vp1)和3c蛋白酶，參考昆蟲(chóng)細(xì)胞的密碼子偏嗜性，在不改變目的蛋白表達(dá)基因的氨基酸序列前提下，對(duì)嵌合p12a和3c基因的密碼子進(jìn)行了優(yōu)化，并在基因兩端分別加上酶切位點(diǎn)，送至公司進(jìn)行了合成。

2、重組轉(zhuǎn)移載體pfastbacdual-p12a3c(o-vp1)的構(gòu)建：

將合成的嵌合基因片段p12a(o-vp1)與桿狀病毒表達(dá)載體pfastbacdual同時(shí)用限制性?xún)?nèi)切酶ecoriandhindiii雙酶切，回收后的p12a(o-vp1)片段和pfastbacdual片段連接后轉(zhuǎn)化top10感受態(tài)細(xì)胞，挑取單克隆菌落后搖菌提取質(zhì)粒，將雙酶切及測(cè)序鑒定為陽(yáng)性的質(zhì)粒命名為pfastbacdual-p12a(o-vp1)。然后將重組質(zhì)粒pfastbacdual-p12a(o-vp1)和3c片段分別用限制性?xún)?nèi)切酶ncoiandkpni雙酶切，回收酶切產(chǎn)物并連接，最后將鑒定正確的質(zhì)粒命名為pfastbacdual-p12a3c(o-vp1)。

3、重組桿狀病毒rbacmid-p12a3c(o-vp1)的構(gòu)建：

將重組轉(zhuǎn)移載體pfastbacdual-p12a3c(o-vp1)轉(zhuǎn)化dh10bac?escherichiacoli感受態(tài)細(xì)胞后進(jìn)行藍(lán)白斑篩選，挑選陽(yáng)性克隆并提取重組桿狀病毒質(zhì)粒rbacmid-p12a3c(o-vp1)，利用通用引物m13f/r進(jìn)行pcr鑒定目的基因是否正確插入。

4、重組桿狀病毒rbac-p12a3c(o-vp1)的構(gòu)建：

重組桿狀病毒質(zhì)粒rbacmid-p12a3c(o-vp1)在cellfectin^?iireagent轉(zhuǎn)染試劑的協(xié)助下轉(zhuǎn)染sf9細(xì)胞。置于27℃細(xì)胞培養(yǎng)箱中培養(yǎng)72小時(shí)，待sf9細(xì)胞出現(xiàn)病變后，將細(xì)胞吹打下來(lái)置于離心管中劇烈振蕩，除去細(xì)胞和碎片收集上清液，即為第一代重組桿狀病毒rbac-dual-p12a3c和rbac-dual。將重組桿狀病毒在sf9細(xì)胞中傳代培養(yǎng)至第三代，保存?zhèn)溆谩?/p>

5、重組蛋白的免疫熒光檢查：

將第三代重組桿狀病毒rbac-p12a3c(o-vp1)病毒液感染sf9細(xì)胞，待細(xì)胞出現(xiàn)病變后，用pbs洗滌后用經(jīng)4%多聚甲醛固定20min、0.5%的tritonx-100穿孔15min及含1%bsa的pbst在37℃下封閉1h；37℃下孵育合適的一抗即o型fmdv兔抗陽(yáng)性血清(1:1000)1h；避光37℃條件下，孵育其對(duì)應(yīng)的二抗即dylight^tm488標(biāo)記的y羊抗兔igg(1:1000)1h；pbs洗滌3次后，于熒光顯微鏡下觀察重組蛋白的間接免疫熒光結(jié)果。

6、重組蛋白的western-blot檢測(cè)：

將重組桿狀病毒rbac-p12a3c(o-vp1)感染sf9細(xì)胞后出現(xiàn)病變，收集細(xì)胞用裂解液裂解，經(jīng)sds-page電泳后轉(zhuǎn)至nc膜，用5%脫脂奶粉封閉1h。孵育相應(yīng)的o型fmdv高免兔抗血清一抗(按1：1000的比例稀釋)，pbst洗滌3次后，然后孵育其對(duì)應(yīng)的hrp標(biāo)記的山羊抗兔igg二抗，最后加ecl顯色液，壓片、曝光。

7、嵌合vlps的電鏡觀察：

將蔗糖密度梯度離心方法收集的目的蛋白，經(jīng)磷鎢酸負(fù)染色處理，然后置于電子顯微鏡下觀察vlps的組裝。

8、口蹄疫病毒嵌合vlps疫苗的制備、豚鼠免疫及免疫原性分析：

將通過(guò)蔗糖密度梯度離心粗提的vlps蛋白與等體積的佐劑isa206乳化成疫苗，商品化的o型口蹄疫滅活疫苗和pbs分別做為陽(yáng)性和陰性對(duì)照組免疫豚鼠。每組5只豚鼠，首免后第21天進(jìn)行二次加強(qiáng)免疫。分別在首免后第7、14、21及28天采集血清進(jìn)行elisa檢測(cè)其體液免疫水平，同時(shí)檢測(cè)利用t細(xì)胞增殖實(shí)驗(yàn)及細(xì)胞因子抗體芯片檢測(cè)其細(xì)胞免疫水平。

9、豚鼠攻毒保護(hù)試驗(yàn)：

在二免后第7天，用100gpid50劑量的口蹄疫病毒o/by/cha/2010的豚鼠毒液對(duì)豚鼠進(jìn)行攻毒，一般選擇后腿皮下攻毒，連續(xù)觀察7d，統(tǒng)計(jì)嵌合vlps疫苗的免疫保護(hù)效率。

攻毒后豚鼠保護(hù)效果結(jié)果如表1所示。

表1

最后應(yīng)說(shuō)明的是：以上所述僅為本發(fā)明的優(yōu)選實(shí)施例而已，并不用于限制本發(fā)明，盡管參照前述實(shí)施例對(duì)本發(fā)明進(jìn)行了詳細(xì)的說(shuō)明，對(duì)于本領(lǐng)域的技術(shù)人員來(lái)說(shuō)，其依然可以對(duì)前述各實(shí)施例所記載的技術(shù)方案進(jìn)行修改，或者對(duì)其中部分技術(shù)特征進(jìn)行等同替換。凡在本發(fā)明的精神和原則之內(nèi)，所作的任何修改、等同替換、改進(jìn)等，均應(yīng)包含在本發(fā)明的保護(hù)范圍之內(nèi)。

序列表

<110>中國(guó)農(nóng)業(yè)科學(xué)院蘭州獸醫(yī)研究所

<120>一種口蹄疫病毒嵌合病毒樣顆粒及其制備方法

<210>1

<211>2259

<212>prt

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glycyscyscysalacysalathrcysalacysalaglythrcyscys

725730735

cysalathralacyscysthrthrglyglythrglythrglyalaala

740745750

cysalaglyalathralacysglyalacyscysalaglythralacys

755760765

alaalaglyalaalaglycysalacysalaalaglycyscysthrthr

770775780

glyglyalacysglycysthrglyglythrcysglythrglyalathr

785790795800

glyglythrglyglythrglythrcysalacyscysglycysthrcys

805810815

alacysalaglythrthralaalacysalaalacysalaglythrgly

820825830

glythrglycysglyglyalaalacysalaglyalathrcysalaala

835840845

glyglythrcysthralacysglycyscysalaalacysalathrcys

850855860

glycyscyscyscysglyalacyscysthralacysglythrcyscys

865870875880

alacysglythrglyglycysthrglyglythrglyalaalacysthr

885890895

cyscyscyscysthrcysglyalaalaglyglyalaglyglyglygly

900905910

alathrthrglythrcyscyscysglyglythrthrglycysalathr

915920925

glythrthrcysglyglyalathrglyglythrthralathrglygly

930935940

cysglyglycysthrthrglyglythrglyalacysglyalacysgly

945950955960

glyalacyscyscysglyalaalaglyalacysalaglycysthrgly

965970975

alacyscyscyscysalathrthrthralacysglyglythralathr

980985990

glyglythrglythralacysalaalacyscyscysglycyscyscys

99510001005

alaglyglyalacyscysalaalacysthralacyscyscyscys

101010151020

glyglyglycysglyglythrthrthralacysalaalaalathr

102510301035

cysthrglythrthrglyglyalacysglythrglyglycysala

104010451050

glyalaglyglycyscysthrglycyscyscyscysalacyscys

105510601065

thrthrthrcysthrthrthrglythrthrthrcysglyalacys

107010751080

glyalathrglyglyglyalaalaalacyscysalathralacys

108510901095

glythrthrglythrglyalacysglyalaglyalaglycysgly

110011051110

glyalacysglyglyalacysalaglycysglycyscysthrcys

111511201125

cysthrglyglycyscysalaalaglythrthrthrglyalacys

113011351140

glythrcysthrcyscyscysthrthrglycysthrglycysala

114511501155

alaalaglycysalacysalathrglythrcysalaalaalacys

116011651170

alacyscysthralacyscysthrthrthrcysalaglyglygly

117511801185

alathralaglycyscyscysalaglythralacysthralacys

119011951200

glycysalacysalaglythralacysthrcysthrglyglycys

120512101215

alacyscysalathrcysalaalacyscysthrglycysalacys

122012251230

thrthrcysalathrglythrthrcysalacysthrglyglycys

123512401245

cyscyscysalacysthrglyalathrthrcysalaalaalaala

125012551260

glycyscyscysglycysthralacysalathrglyglythrthr

126512701275

glycysalathralathrglythrcyscyscysglycyscyscys

128012851290

glyglycysalathrglyglyalaglyalacysglycyscysgly

129513001305

cyscysglyglyalacysalacysalacyscysthrglyalagly

131013151320

glyalaalaglycysthrglycysthrcysalacysthrglythr

132513301335

alathrcyscysalacysglycyscysglyalaglythrglygly

134013451350

glyalacysalacysalaglyglyalacysthrglyalaalacys

135513601365

thrcyscysalaalaalathrthrcysalacysthrthrthrthr

137013751380

thrcysthralathrcyscyscysglythralacysglythrgly

138513901395

thrcysthrglycyscysglycysalaglyalathrthralacys

140014051410

glycysalathralacysalacyscysglycysglythrcysthr

141514201425

glyalacysglythrglyglycysalaglyalaglyalacysala

143014351440

alacysalaalaalacysglythralacysalaglyglyglyala

144514501455

thrglyglyglythrcysthrglycysalathrcysthralacys

146014651470

cysalaglyalathrcysalacysalacysalacysglyglygly

147514801485

alaalaglyglycysthrglyalaalaglyalacysglyalacys

149014951500

alacysthrthrthrglyglythrcysglythrglythrcysgly

150515101515

cysthrthralaglycysglycysthrglyglycysalaalagly

152015251530

glyalathrthrthrthrglyalaalacysthrglycysglycys

153515401545

cysthrcyscyscysglyalathrthrglyalacyscyscyscys

155015551560

cysglycysthrcysalacysalaalaalacyscysalacysthr

156515701575

thrcysglyalacysalaglyglycysglyalaglythrcysgly

158015851590

glycysthrglyalacyscyscyscysglythrglyalacysthr

159516001605

glycyscysalacyscysglythrthrglyalaglyalaalacys

161016151620

thralacysglyglycysglyglycysglyalaglyalacysala

162516301635

cysalaalaglythrcyscysalaglyalaglyglycysglycys

164016451650

cysalacyscysalacysalacysalaglyalacysglythrcys

165516601665

thrcysalathrthrcysalathralathrthrglyglyalacys

167016751680

alaglyalathrthrthrglythrglyalaalaalaglythrcys

168516901695

alacysalacyscysalaalaalaalaglyalacysthrcysala

170017051710

alathralaalaalathrglythralathrthrglyglyalacys

171517201725

cysthrglyalathrglycysalaglyalacyscyscyscyscys

173017351740

cyscyscyscysalacysalacyscyscysthralaglythrala

174517501755

glyglyglyglycysglycysthrcyscysthrcyscysglycys

176017651770

alacysthrglycyscysalacysthrthralacysthralathr

177517801785

thrthrcysglycysthrglyalathrcysthralaglyalagly

179017951800

glythrglyglycysalaglythrglyalaalaalacysalacys

180518101815

alaalaglyglyglyglyglyalacyscysthrthralacyscys

182018251830

thrglyglyglythrglycyscysalaalaalathrglyglyala

183518401845

glycysalacyscysthrglyalaalaglycysalaglycyscys

185018551860

thrthrglyglyalacysalaalacysalacyscysalacyscys

186518701875

alaalacyscyscysalaalacysglyglycysglythralacys

188018851890

cysalathralaalaglyglycysglycyscysglycysthrthr

189519001905

alacyscyscysglyglycysthrthrglycysalathrthrgly

191019151920

cyscyscysthralacysalacysglyglycysalacyscysala

192519301935

cysalacyscysglythrglythrthrthrthrglyglycyscys

194019451950

alacyscysglythrthrthralacysalaalacysglyglygly

195519601965

alaalacysthrglycysalaalaalathralacysglycyscys

197019751980

glyglyglyglyglycysthrcysalacysthrglycyscyscys

198519901995

alaalacysglythrglyalaglyalaglyglythrglyalathr

200020052010

cysthrcyscysalaalaglythrglycysthrglyglycysthr

201520202025

cysalaglyalaalaglyglycysglyglycysglythrglygly

203020352040

cyscysglycysthrglycyscysthralacysthrthrcysthr

204520502055

thrthrcysalaalacysthralacysglyglythrglycyscys

206020652070

alathrcysalaalaalaglycyscysalacysthrcysglygly

207520802085

glythrglyalacysalaglyalaalacysthrglycysthrgly

209020952100

thralacyscysglycysalathrglyalaalaglyalaglygly

210521102115

glycyscysglyalaglyalacysglythralacysthrglythr

212021252130

cyscysthrcysglyglycyscyscyscysthrcysthrthrgly

213521402145

glycysthrglythrthrcysalacyscyscysglyalaglythr

215021552160

glycysglyglycysthralaglyalacysalacysalaalaala

216521702175

cysalaglyalaalaalaalathralaglythrglyglycysala

218021852190

cyscysthrglythralaalaalaglycysalaglythrcyscys

219522002205

thrthrglyalaalacysthrthrthrglyalacyscysthrgly

221022152220

cysthrcysalaalaglythrthrglyglycysalaglyglyala

222522302235

glyalacysglythrglyglyalaglythrcyscysalaalacys

224022452250

cyscysthrglyglygly

2255

<210>1

<211>2259

<212>dna

<213>嵌合衣殼蛋白p12a-3c的dna序列

<400>2

ggcgccgggcaatccagcccggccaccggctcgcagaatcaatctggcaacactggaagc60

atcattaacaactactacatgcagcagtaccagaactcaatggacacacaacttggtgac120

aacgccatcagtggaggttccaatgagggttccactgatacaacctccacacacacaaac180

aacacccagaacaatgactggttttcaaagcttgcaagttctgccttcaccggtctcttc240

ggcgcactgctcgctgacaagaagacagaggagactactcttctggaagaccgcatcctc300

actacccgcaatgggcacaccacctccacgacacagtcgagtgtgggagtcacatacggg360

tactccaccggggaagaccacgtttccgggcctaacacatctggcttggagacgcgggtg420

acacaggcagagagattcttcaagaaacacctgtttgattggacaacggacaaagctttt480

ggacacttggaaaagctggaactccccactgaccacaagggtgtctacggacacctggtg540

gactcatttgcatacatgagaaatggttgggacgtggaggtgtccgctgttggcaaccag600

ttcaacggcgggtgtctcctggtggccatggtccctgaatacaaagacttcactctgcgt660

gagaagtaccagcttaccctgttcccgcaccagttcattagccccagaaccaacatgact720

gcccacatcacagtcccataccttggtgtgaacagatacgaccagtacaagaagcacaag780

ccttggacgctggtcgtgatggtggtgtcaccgctcacagttaacaacagtggtgcggaa840

cagatcaaggtctacgccaacatcgccccgacctacgtccacgtggctggtgaactcccc900

tcgaaggaggggattgtcccggttgcatgttcggatggttatggcggcttggtgacgacg960

gacccgaagacagctgaccccatttacggtatggtgtacaacccgcccaggaccaactac1020

cccgggcggtttacaaatctgttggacgtggcagaggcctgccccacctttctttgtttc1080

gacgatgggaaaccatacgttgtgacgagagcggacggacagcgcctcctggccaagttt1140

gacgtctcccttgctgcaaagcacatgtcaaacacctacctttcagggatagcccagtac1200

tacgcacagtactctggcaccatcaacctgcacttcatgttcactggccccactgattca1260

aaagcccgctacatggttgcatatgtcccgcccggcatggagacgccgccggacacacct1320

gaggaagctgctcactgtatccacgccgagtgggacacaggactgaactccaaattcact1380

ttttctatcccgtacgtgtctgccgcagattacgcatacaccgcgtctgacgtggcagag1440

acaacaaacgtacagggatgggtctgcatctaccagatcacacacgggaaggctgaagac1500

gacactttggtcgtgtcgcttagcgctggcaaggattttgaactgcgcctcccgattgac1560

ccccgctcacaaaccacttcgacaggcgagtcggctgaccccgtgactgccaccgttgag1620

aactacggcggcgagacacaagtccagaggcgccaccacacagacgtctcattcatattg1680

gacagatttgtgaaagtcacaccaaaagactcaataaatgtattggacctgatgcagacc1740

cccccccacaccctagtaggggcgctcctccgcactgccacttactatttcgctgatcta1800

gaggtggcagtgaaacacaagggggaccttacctgggtgccaaatggagcacctgaagca1860

gccttggacaacaccaccaacccaacggcgtaccataaggcgccgcttacccggcttgca1920

ttgccctacacggcaccacaccgtgttttggccaccgtttacaacgggaactgcaaatac1980

gccgggggctcactgcccaacgtgagaggtgatctccaagtgctggctcagaaggcggcg2040

tggccgctgcctacttctttcaactacggtgccatcaaagccactcgggtgacagaactg2100

ctgtaccgcatgaagagggccgagacgtactgtcctcggcccctcttggctgttcacccg2160

agtgcggctagacacaaacagaaaatagtggcacctgtaaagcagtccttgaactttgac2220

ctgctcaagttggcaggagacgtggagtccaaccctggg2259

<210>1

<211>639

<212>prt

<213>3c蛋白氨基酸序列

<400>3

alaglythrglyglythrglycyscyscyscysalacyscysglyala

151015

cyscysglyalacysthrthrglycysalaalaalaalaglyalathr

202530

glyglythrcysalathrglyglyglycysalaalacysalacysthr

354045

alaalaglycyscysthrglythrthrglyalaglycysthrcysala

505560

thrthrcysthrcysglyalacysglyglyglyalaalaglyalacys

65707580

glyglythralaglycyscysalathrcysthrglycysthrglythr

859095

glycysthralacyscysglyglyalaglythrglythrthrthrgly

100105110

glycysalacysthrglycyscysthralacyscysthrthrglythr

115120125

alacyscysthrcysglythrcysalacyscysthrcysthrthrcys

130135140

glycysglyglyalaglyalaalaglythralacysglyalacysala

145150155160

alaglyalathrcysalathrglycysthrglyglyalacysglygly

165170175

cysalaglyalaglycysthralathrglyalacysalaglyalacys

180185190

alaglythrglyalacysthralacysalaglyalaglythrglythr

195200205

thrthrglyalaglythrthrthrglyalaglyalathrthralaala

210215220

alaglythralaalaalaalaglyglyalacysalaglyglyalacys

225230235240

alathrglycysthrcysthrcysalaglyalacysglycyscysgly

245250255

cysalacysthrcysalathrglyglythrglycysthrthrcysala

260265270

cyscysglythrglyglyglyalaalathrcysglycysglythrgly

275280285

alaglyglyglyalacysalathrcysalacysglyalaalaalacys

290295300

alacysthrthrthrcysglythrglyalathralacysalaglycys

305310315320

alaalaglyalaalathrglyalaalaglyalaalaalaglyglycys

325330335

alacyscyscyscyscysglythrcysglythrcysglyglycysgly

340345350

thrglyalathrcysalaalacysalaalacysglycyscysglyala

355360365

thrglythrthrglyglyglyalaglyalacysthrglyalathrthr

370375380

thrthrcysthrcysthrglyglythrglyalaglyglycyscyscys

385390395400

thrthralacyscysthralacysalaalaglyglyalacysalathr

405410415

thrglythralaglythrglythrglycysalathrglyglyalathr

420425430

glyglyalaglyalacysalacyscysalathrglycyscyscysgly

435440445

glycyscysthrcysthrthrthrglycyscysthralacysalaala

450455460

alaglycyscysglycyscysalacyscysalaalaglyglycysthr

465470475480

glyglycysthralacysthrglythrglyglyalaglyglyalagly

485490495

cyscysglythrthrcysthrthrglycysalaalaalaglyglyala

500505510

cysglyglyalaglycysthrglyalacysalacysalathrthrcys

515520525

alathrcysglythrcysglyglycysalacysthrcysalacysthr

530535540

cysthrglycysalaglyglythrglyglycysalaalathrglygly

545550555560

alaglythrthrglyglyalathralathrthrglycysthrcysala

565570575

thrglycysglythrthrthrcyscysalaglyglythrcyscysala

580585590

thrglycysthrthrcysthrthralaalaalaalathrglyalaala

595600605

glyglycysalacysalacysalathrcysglyalacyscyscyscys

610615620

glyalaalacyscysalacysalacyscysalacysglyalagly

625630635

<210>1

<211>639

<212>dna

<213>3c蛋白的dna序列

<400>4

agtggtgccccaccgaccgacttgcaaaagatggtcatgggcaacactaagcctgttgag60

ctcattctcgacgggaagacggtagccatctgctgtgctaccggagtgtttggcactgcc120

taccttgtacctcgtcacctcttcgcggagaagtacgacaagatcatgctggacggcaga180

gctatgacagacagtgactacagagtgtttgagtttgagattaaagtaaaaggacaggac240

atgctctcagacgccgcactcatggtgcttcaccgtgggaatcgcgtgagggacatcacg300

aaacactttcgtgatacagcaagaatgaagaaaggcacccccgtcgtcggcgtgatcaac360

aacgccgatgttgggagactgattttctctggtgaggcccttacctacaaggacattgta420

gtgtgcatggatggagacaccatgcccggcctctttgcctacaaagccgccaccaaggct480

ggctactgtggaggagccgttcttgcaaaggacggagctgacacattcatcgtcggcact540

cactctgcaggtggcaatggagttggatattgctcatgcgtttccaggtccatgcttctt600

aaaatgaaggcacacatcgaccccgaaccacaccacgag639