CN103740736B - 化学合成的HSV2病毒gE糖蛋白胞外区基因片段及其表达、应用 - Google Patents
化学合成的HSV2病毒gE糖蛋白胞外区基因片段及其表达、应用 Download PDFInfo
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Abstract
本发明化学合成的HSV2病毒gE糖蛋白胞外区基因片段及其表达、应用涉及基因工程技术、疫苗和诊断试剂领域。本发明是通过计算机分析，筛选出HSV2病毒gE糖蛋白内的强抗原表位，第21个氨基酸至第414个氨基酸，共394个氨基酸，选择真核和原核生物均偏爱的密码子，化学合成抗原表位的全新基因序列，利用基因工程技术，表达该基因片段、制备HSV2病毒gE糖蛋白的强抗原表位片段。表达的HSV2病毒gE糖蛋白的强抗原表位片段，可用于HSV2疫苗研制、抗体的检测及用于免疫制备抗HSV2病毒单抗和多抗等。
Description
技术领域
本发明涉及的是化学合成的HSV2病毒gE糖蛋白胞外区的全新基因片段，利用基因工程技术，制备重组HSV2病毒gE糖蛋白。通过计算机分析，筛选出含强抗原表位的HSV2病毒的gE糖蛋白胞外区片段，选择真核和原核生物均偏爱的密码子，化学合成全新的基因序列，利用基因工程技术表达，表达的蛋白可用于疫苗及HSV2病毒抗体或抗原的检测等，本发明涉及基因工程技术、疫苗和诊断试剂领域。
背景技术
单纯疱疹病毒（Herpers simplex virus, HSV）属于疱疹病毒科α亚科，是人类感染的常见病毒之一，根据血清型可分为I和II型两种。其中，I型主要通过口面部感染，也存在性传播，II型主要通过性传播，并且和女性***的发生密切相关。近期越来越多的研究表明，HSV I对阿兹海默症的发生有关联，HIV的感染和HSV II也有联系。HSV在全球普遍流行，HSV I型在成人血清中阳性率高达85%以上，HSV II型全球血清阳性率约为20%，并且发病人数以每年25%速度增加。
HSV可在感染组织或附近部位繁殖形成原发病灶，并在三叉神经核脊神经建立终身的潜伏感染，当机体受到外界刺激或免疫力下降是，HSV病毒下行至表皮大量增殖，形成复发感染。HSV反复发作与HSV免疫逃逸相关，HSV通过躲避人体免疫***的攻击实现潜伏感染和复发感染，因此HSV一旦感染，很难被清除，患者受到长期疾病折磨。
病毒糖蛋白 (glycoproteins, gp) 是病毒毒粒表面的抗原决定簇，目前已发现并正式命名的包膜糖蛋白共有12个(gB、gC、gD、gE、gG、gH、gI、gK、gL和gM等)。gB和gD糖蛋白参与病毒与宿主细胞的吸附和融合，已被选作候选疫苗，对其研究较多。近年研究发现，gE2是免疫逃逸相关的包膜糖蛋白。HSV病毒包膜蛋白gE与IgG Fc端结合，从而阻断多个补体介导的免疫途径，使HSV病毒成功躲避机体的免疫***攻击。因此他是造成病毒潜伏感染的蛋白分子之一。选取gE糖蛋白用于免疫机体，阻断病毒的潜伏感染，有希望成为理想的候选疫苗。
本发明通过计算机分析，筛选出含强抗原表位的HSV2病毒的gE糖蛋白胞外区片段，选择真核和原核生物均偏爱的密码子，化学合成全新的基因序列，利用基因工程技术表达，表达产物具有较好的抗原性和特异性，表达的蛋白可用于疫苗及HSV2病毒抗体或抗原的检测等。
发明内容
本发明是化学合成的HSV2病毒gE糖蛋白胞外区的全新基因片段，利用基因工程技术，制备重组HSV2病毒gE糖蛋白胞外区片段。通过计算机分析，筛选出含强抗原表位的HSV2病毒gE糖蛋白胞外区片段,第21个氨基酸 — 第414个 氨基酸，共394个氨基酸，选择真核和原核生物均偏爱的密码子，化学合成全新的基因序列，利用基因工程技术表达该基因。表达的蛋白可用于疫苗、HSV2病毒抗体或抗原的检测及用于免疫制备抗HSV2病毒单抗和多抗等。
本发明通过计算机分析，筛选出含强抗原表位的HSV2病毒的gE糖蛋白胞外区片段，选择真核和原核生物均偏爱的密码子，化学合成全新的基因序列，利用基因工程技术表达，表达产物具有较好的抗原性和特异性，表达的蛋白可用于疫苗及HSV2病毒抗体或抗原的检测等。
化学合成的HSV2病毒gE糖蛋白胞外区基因片段及其表达、应用是采取以下步骤实现的：
1．HSV2病毒gE糖蛋白胞外区抗原表位的筛选及其基因片段的化学合成：
利用ANTHEWIN等软件，通过计算机分析HSV2病毒gE糖蛋白的氨基酸序列， 发现gE糖蛋白的N端（第21氨基酸 — 第414 氨基酸）含有较强的抗原决定簇。选择真核和原核生物均偏爱的密码子，化学合成全新的基因序列，并且在5'端增加了EcoRI酶切位点(下划线部分)、起始密码子（ATG）、信号肽（GGCTGGAGCTGTATTATCCTGTTCCTGGTCGCCACTGCCACTGGAGTTCATTCTGCC），在3'端增加了His标记（CAC CAC CAC CAC CAC CAC）、终止密码子（TGA）和Himd III酶切位点(下划线部分)，使该基因片段易于克隆至质粒pMCE5内的EcoRI和Himd III酶切位点内。
筛选的HSV2病毒gE糖蛋白内的抗原表位氨基酸序列（第21个aa － 第414个aa）：
Ala Ala Pro Arg Thr Ser Trp Lys Arg Val Thr Ser Gly Glu Asp Val ValLeu Leu Pro Ala Pro Ala Glu Arg Thr Arg Ala His Lys Leu Leu Trp Ala Ala GluPro Leu Asp Ala Cys Gly Pro Leu Arg Pro Ser Trp Val Ala Leu Trp Pro Pro ArgArg Val Leu Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Glu Pro Leu AlaIle Ala Tyr Ser Pro Pro Phe Pro Ala Gly Asp Glu Gly Leu Tyr Ser Glu Leu AlaTrp Arg Asp Arg Val Ala Val Val Asn Glu Ser Leu Val Ile Tyr Gly Ala Leu GluThr Asp Ser Gly Leu Tyr Thr Leu Ser Val Val Gly Leu Ser Asp Glu Ala Arg GlnVal Ala Ser Val Val Leu Val Val Glu Pro Ala Pro Val Pro Thr Pro Thr Pro AspAsp Tyr Asp Glu Glu Asp Asp Ala Gly Val Thr Asn Ala Arg Arg Ser Ala Phe ProPro Gln Pro Pro Pro Arg Arg Pro Pro Val Ala Pro Pro Thr His Pro Arg Val IlePro Glu Val Ser His Val Arg Gly Val Thr Val His Met Glu Thr Leu Glu Ala IleLeu Phe Ala Pro Gly Glu Thr Phe Gly Thr Asn Val Ser Ile His Ala Ile Ala HisAsp Asp Gly Pro Tyr Ala Met Asp Val Val Trp Met Arg Phe Asp Val Pro Ser SerCys Ala Asp Met Arg Ile Tyr Glu Ala Cys Leu Tyr His Pro Gln Leu Pro Glu CysLeu Ser Pro Ala Asp Ala Pro Cys Ala Val Ser Ser Trp Ala Tyr Arg Leu Ala ValArg Ser Tyr Ala Gly Cys Ser Arg Thr Thr Pro Pro Pro Arg Cys Phe Ala Glu AlaArg Met Glu Pro Val Pro Gly Leu Ala Trp Leu Ala Ser Thr Val Asn Leu Glu PheGln His Ala Ser Pro Gln His Ala Gly Leu Tyr Leu Cys Val Val Tyr Val Asp AspHis Ile His Ala Trp Gly His Met Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn AlaVal Val Glu Gln His Leu Pro Gln Arg Gln Pro Glu Pro Val Glu Pro Thr Arg ProHis Val Arg Ala Pro His Pro Ala Pro Ser Ala Arg Gly Pro Leu Arg
化学合成的含HSV2病毒gE糖蛋白胞外区抗原表位基因的DNA 序列（1282 bp）：
GAATTC GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCCACT GCC ACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGCGGA GAG GAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTGCTG TGG GCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCCCTG TGG CCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCTCCC GAA CCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTGTAT AGT GAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATCTAC GGC GCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCCGAC GAG GCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCTACT CCA ACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGACGC TCA GCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACCCAC CCA CGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAGACC CTG GAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATCCAC GCT ATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTTGAT GTG CCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCTCAG CTG CCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCCTAC CGA CTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGGTGT TTC GCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACAGTG AAC CTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTGGTC TAT GTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCACAG TAC AGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTGGAA CCA ACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCCCTG CGT CAC CAC CAC CAC CAC CAT TGA TAAGCTT
2．表达HSV2病毒gE糖蛋白胞外区片段重组质粒的构建:
提取质粒pMCE5,用EcoR I和Hind III双酶切，电泳后回收酶切的质粒大片段, 溶于去离子水内。同样用EcoR I和Hind III双酶切化学合成的HSV2病毒gE糖蛋白基因片段，电泳回收后, 溶于去离子水内。
取等摩尔浓度的上述两种酶切后DNA片段，在同一离心管内用T4 DNA 连接酶连接，使HSV2病毒gE糖蛋白基因片段***到载体pMCE5内的EcoR I和Hind III位点之间，与载体上的起始密码子翻译框架一致，表达一个融合蛋白。
3．重组质粒的筛选与鉴定:
将重组质粒转化大肠杆菌DH5α，涂布含100μg／ml氨苄青霉素的LB平板，置37℃ 过夜。次日随机挑取转化菌落，含质粒pMCE5转化菌作阴性对照，含质粒pUC57-gC21转化菌作阳性对照，菌液PCR扩增HSV2病毒gE糖蛋白胞外区基因片段,含HSV2病毒gE糖蛋白胞外区基因片段的阳性重组质粒，应扩增出长约1282bp 的基因片段。在提取阳性单菌落质粒，进行双酶切鉴定并将含有外源基因的质粒进行DNA序列分析，序列分析证实重组质粒含有合成的HSV2病毒gE糖蛋白基因片段，序列完全正确：
GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCC ACT GCCACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGC GGA GAGGAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTG CTG TGGGCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCC CTG TGGCCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCT CCC GAACCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTG TAT AGTGAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATC TAC GGCGCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCC GAC GAGGCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCT ACT CCAACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGA CGC TCAGCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACC CAC CCACGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAG ACC CTGGAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATC CAC GCTATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTT GAT GTGCCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCT CAG CTGCCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCC TAC CGACTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGG TGT TTCGCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACA GTG AACCTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTG GTC TATGTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCA CAG TACAGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTG GAA CCAACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCC CTG CGTCAC CAC CAC CAC CAC CAT TGA T
构建的重组质粒表达HSV2的病毒gE糖蛋白胞外区片段（394个氨基酸），在其N端融合了载体上的19个氨基酸，在其C端融合了载体上的6个氨基酸，全长419个氨基酸,其氨基酸序列如下：
N端：Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr GlyVal His Ser
C端：His His His His His His
4．重组质粒在CHO细胞中的表达鉴定：
CHO细胞在不含血清的细胞表达培养基在锥形瓶中震荡培养，37℃，5% CO2。转染前一天，以适当的浓度将CHO细胞接种于不含血清的细胞表达培养基中，震荡悬浮培养，37℃，5% CO2。用DNA和PEI混合物转染重组质粒。转染后第五天收集细胞上清，检测蛋白表达水平，第六天收集所有上清用于纯化重组蛋白。
5．重组蛋白的纯化、鉴定和浓度检测：
收集细胞上清100ml，离心后将上清以1ml/min的速度加入His TrapTM FF crudecolumn中。10倍体积平衡液（1×PBS，0.5mol/L NaCl，20mmol/L imidazole，pH7.4）洗脱杂蛋白，再加入1ml洗脱液（1×PBS，0.5mol/L NaCl，500mmol/L imidazole，pH7.4）洗脱目的蛋白，静置20分钟后洗脱，收集洗脱液，用透析液（1×PBS，0.5mol/L NaCl，pH7.4）透析过夜，即为纯化的目的蛋白，SDS-PAGE、Western Blot检测，并测定蛋白浓度。
6．免疫小鼠及gE2抗血清的制备：
10只雄性昆明小鼠随机分为2组，每组5只。实验组以纯化的重组gE2糖蛋白与弗氏佐剂等体积混合，充分乳化，分别于第0，2，4周，腹腔注射免疫小鼠，2 ug/只/次，0.1ml/只，对照组以PBS等体积混合弗氏佐剂，充分乳化，0.1ml/只。每次免疫后2周眼眶采血。血液37℃放置1小时后4℃过夜，4000rpm离心10分钟，取上清即得重组蛋白gE2抗血清。
7. 重组蛋白gE2的免疫原性检测：
用1×CBS将纯化的重组gE2糖蛋白稀释为1 ug/ml，包被酶联板，每孔100ul，4℃过夜。次日5%FBS封闭酶联板，每孔130ul, 室温2小时。将制备的抗血清用样本稀释液按1:500，1:2500，1:12500逐级稀释后，分别加至封闭后的酶联板孔内，每孔100ul，37℃反应1小时，用PBST洗5遍后，加1:5000稀释的羊抗小鼠IgG-HRP，每孔100ul，37℃反应30 分钟，PBST洗5遍，加底物TMB溶液每孔100 ul，37℃避光显色10 分钟，加50ul 1N盐酸终止反应，用酶联仪测定A450值。结果判定，以待测样品A值与阴性对照A值之比大于或等于2.1（P/N≥2.1）且A值≥0.15为阳性，显示阳性结果最大的抗体稀释度为免疫小鼠的血清抗体滴度。数据用均数±标准差()表示，进行统计学分析，用GraphPad Prism 5.0、SPSS软件进行统计学分析，认为P＜0.05为有显著性差异。
所述的化学合成的HSV2病毒gE糖蛋白胞外区的基因片段序列，利用细菌、酵母细胞、昆虫细胞、哺乳动物细胞及转基因动植物进行重组表达、制备。
上述所述的方法制备的HSV2病毒gE糖蛋白胞外区片段，用于HSV2病毒亚单位疫苗的研制及抗体检测。
本发明与现有技术相比具有的优点
本发明表达的HSV2病毒的gE糖蛋白片段，有较多优点:
1．目前应用的HSV2病毒抗体的酶联免疫检测试剂盒，其使用的抗原是全病毒抗原，生产较危险、成本高、与其它病毒有交叉反应等缺点。表达的HSV2病毒的gE糖蛋白胞外区片段用作抗原可克服上述缺点。
2．目前缺乏HSV2病毒疫苗。灭活疫苗的研究取得了较大进展，但是生产成本高、危险大。HSV2病毒的gE糖蛋白在感染的细胞中含量最多,是疱疹病毒家族中高度保守的蛋白,各毒株间差异较小,同时gE蛋白具有较强的免疫原性,可诱导机体产生中和抗体和细胞免疫反应,并且和病毒逃逸机制相关,因此HSV2-gE糖蛋白是构建HSV疫苗理想的目的基因。我们选择了其强抗原表位，利用基因工程技术表达制备，为研制基因工程疫苗奠定基础。基因工程疫苗安全、成本低。
3．根据筛选出的HSV2病毒的gE糖蛋白胞外区片段氨基酸序列，选择真核和原核生物均偏爱的密码子，化学合成全新的基因序列，该基因适宜在真核和原核细胞内高表达。
4．本发明构建的表达HSV2病毒的gE糖蛋白的CHO细胞株，可大量纯化制备该蛋白，易于纯化，且具有相当强的免疫原性。目前未见表达该段蛋白的报道。
附图说明
以下将结合附图对本发明作进一步说明：
图1是菌液PCR检测8个重组子的PCR扩增产物。1：2000 DL DNA（TaKaRa）；2：gE2基因PCR产物；3-10：1-8号挑取单菌落菌液PCR产物； 11： 空pMCE5/DH5α菌液PCR产物。
图2是双酶切鉴定结果。 1：5000 DL DNA（TaKaRa）；2： 重组质粒经EcoRI 和HindIII双酶切产物； 3： 重组质粒经EcoRI 单酶切产物；4：gE2基因。
图3是表达HSV2病毒的gE糖蛋白重组菌的SDS-PAGE分析结果。 M： ProteinMarker； 1： 还原条件下电泳； 2： 非还原条件电泳
图4是表达HSV2病毒的gE糖蛋白纯化前后的Western-Blots分析结果。M： ProteinMarker； 1： 还原条件下电泳； 2： 非还原条件电泳；P： Multiple-tag作为阳参。
图5是ELISA检测重组蛋白gE2免疫原性。
具体实施方式
本发明实施方式的详细说明：
HSV2病毒的gE糖蛋白抗原表位的分析、基因合成及表达
通过计算机分析HSV2病毒的gE糖蛋白的全部氨基酸序列，筛选出HSV2病毒的gE糖蛋白内的强抗原表位,选用真核偏爱的密码子，化学合成HSV2病毒的gE糖蛋白强抗原表位的全新基因片段。将基因片段克隆至质粒pMCE5内的EcoRI/HindIII位点，与载体上的起始密码子的翻译框架一致，可表达一个融合蛋白。将构建的真核表达质粒pMCE5-gE2并转染至CHO细胞进行真核表达，筛选获得了高表达gE糖蛋白的细胞株。
材料与方法
1. 菌种与质粒: 宿主菌DH5α及表达载体pMCE5为本实验室保存。
2. 分子生物学试剂:限制性内切酶EcoRI、HindIII、及T4 DNA连接酶为TaKaRa公司产品。质粒纯化试剂盒及从琼脂糖凝胶内回收DNA片段的试剂盒为TaKaRa公司产品.其它试剂为进口或国产分析纯试剂。
3. 基因片段的合成: 由大连TaKaRa公司帮助合成。
4. 基因克隆方法: DNA的酶切、连接、电泳；质粒的提取、转化。其它试剂盒按说明书进行操作。
5. DNA序列分析：用TAKARA公司质粒纯化试剂盒纯化质粒，用DNA全自动测序仪测序。
结果
1. HSV2病毒的gE糖蛋白抗原表位筛选及基因片段的合成：
利用ANTHEWIN等软件，通过计算机分析HSV2病毒的gE糖蛋白的全部氨基酸序列（UniProtKB: P89475.1），筛选出HSV2病毒的gE糖蛋白内的强抗原表位,即从第21个氨基酸到第414个氨基酸，其氨基酸序列如下：
Ala Ala Pro Arg Thr Ser Trp Lys Arg Val Thr Ser Gly Glu Asp Val ValLeu Leu Pro Ala Pro Ala Glu Arg Thr Arg Ala His Lys Leu Leu Trp Ala Ala GluPro Leu Asp Ala Cys Gly Pro Leu Arg Pro Ser Trp Val Ala Leu Trp Pro Pro ArgArg Val Leu Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Glu Pro Leu AlaIle Ala Tyr Ser Pro Pro Phe Pro Ala Gly Asp Glu Gly Leu Tyr Ser Glu Leu AlaTrp Arg Asp Arg Val Ala Val Val Asn Glu Ser Leu Val Ile Tyr Gly Ala Leu GluThr Asp Ser Gly Leu Tyr Thr Leu Ser Val Val Gly Leu Ser Asp Glu Ala Arg GlnVal Ala Ser Val Val Leu Val Val Glu Pro Ala Pro Val Pro Thr Pro Thr Pro AspAsp Tyr Asp Glu Glu Asp Asp Ala Gly Val Thr Asn Ala Arg Arg Ser Ala Phe ProPro Gln Pro Pro Pro Arg Arg Pro Pro Val Ala Pro Pro Thr His Pro Arg Val IlePro Glu Val Ser His Val Arg Gly Val Thr Val His Met Glu Thr Leu Glu Ala IleLeu Phe Ala Pro Gly Glu Thr Phe Gly Thr Asn Val Ser Ile His Ala Ile Ala HisAsp Asp Gly Pro Tyr Ala Met Asp Val Val Trp Met Arg Phe Asp Val Pro Ser SerCys Ala Asp Met Arg Ile Tyr Glu Ala Cys Leu Tyr His Pro Gln Leu Pro Glu CysLeu Ser Pro Ala Asp Ala Pro Cys Ala Val Ser Ser Trp Ala Tyr Arg Leu Ala ValArg Ser Tyr Ala Gly Cys Ser Arg Thr Thr Pro Pro Pro Arg Cys Phe Ala Glu AlaArg Met Glu Pro Val Pro Gly Leu Ala Trp Leu Ala Ser Thr Val Asn Leu Glu PheGln His Ala Ser Pro Gln His Ala Gly Leu Tyr Leu Cys Val Val Tyr Val Asp AspHis Ile His Ala Trp Gly His Met Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn AlaVal Val Glu Gln His Leu Pro Gln Arg Gln Pro Glu Pro Val Glu Pro Thr Arg ProHis Val Arg Ala Pro His Pro Ala Pro Ser Ala Arg Gly Pro Leu Arg
根据筛选的HSV2病毒gE糖蛋白内的抗原表位氨基酸序列，选择真核的密码子，化学合成全新的基因序列，并且在5'端增加了EcoRI酶切位点(下划线部分)、起始密码子（ATG）、信号肽（GGCTGGAGCTGTATTATCCTGTTCCTGGTCGCCACTGCCACTGGAGTTCATTC TGCC），在3'端增加了His标记（CAC CAC CAC CAC CAC CAC）、终止密码子（TGA）和Himd III酶切位点(下划线部分)，使该基因片段易于克隆至质粒pMCE5内的EcoRI和Hind III酶切位点内。化学合成的含HSV2病毒gE糖蛋白抗原表位基因的DNA 序列（1282 bp）如下：
GAATTC GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCCACT GCC ACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGCGGA GAG GAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTGCTG TGG GCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCCCTG TGG CCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCTCCC GAA CCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTGTAT AGT GAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATCTAC GGC GCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCCGAC GAG GCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCTACT CCA ACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGACGC TCA GCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACCCAC CCA CGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAGACC CTG GAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATCCAC GCT ATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTTGAT GTG CCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCTCAG CTG CCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCCTAC CGA CTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGGTGT TTC GCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACAGTG AAC CTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTGGTC TAT GTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCACAG TAC AGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTGGAA CCA ACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCCCTG CGT CAC CAC CAC CAC CAC CAT TGA TAAGCTT
2．表达HSV2病毒gE糖蛋白胞外区片段重组质粒的构建:
提取质粒pMCE5,用EcoRI和Hind III双酶切，电泳后回收酶切的质粒大片段, 溶于去离子水内。同样用EcoRI和Hind III双酶切化学合成的HSV2病毒gE糖蛋白胞外区基因片段，电泳回收后, 溶于去离子水内。
取等摩尔浓度的上述两种酶切后DNA片段，在同一离心管内用T4 DNA 连接酶连接，使HSV2病毒gE糖蛋白胞外区基因片段***到载体pMCE5内的EcoRI和Hind III位点之间，与载体上的起始密码子翻译框架一致，表达一个融合蛋白。
3．重组质粒的筛选与鉴定:
将上步连接的重组质粒转化到大肠杆菌DH5α,将转化产物涂布含100µg／ml氨苄青霉素的固体LB培养基上，置37℃培养过夜。次日随机挑选1-8号转化子菌落、一个含质粒pMCE5转化菌作阴性对照和一个含质粒pUC57-gE2转化菌作阳性对照，分别接种到3 ml含氨苄青霉素100µg／ml的液体LB培养基， 37℃振荡培养5h,取菌液1ml，离心收菌。分别用50μl去离子水悬浮菌体，沸水煮5min,离心（4℃，12000rpm ）5min, 取上清2μl用作PCR模板,PCR扩增***载体内的HSV2病毒gE糖蛋白胞外区基因片段，PCR 反应浓度为：质粒模板2μl、HSV2病毒gE糖蛋白胞外区基因片段的正链P1（CCGGAATTCGCCGCCACCATGGG）和负链引物P2（AAATGCGGCCGCTGACCATG ATTACGCCAAGCT）各1μl、10×pyrobest buffer 5.0μl、 2.5mmol/L dNTP 4.0μl、Pyrobest DNA Taq 酶0.5μl (2.5 U)、去离子水36.5μl,总体积50μl。扩增条件为：94℃ 30秒、55℃ 30秒、72℃4分钟，35个循环；最后72℃延伸7分钟。 取PCR扩增产物5μl,用1.0％的Agarose凝胶检测, 结果，1号转化子扩增出1282bp的目的基因片段（见图1），而含质粒pMCE5的对照菌没有扩增出该基因片段。初步证实，这1号转化子含有HSV2病毒gE糖蛋白基因片段。
提取3号重组子的质粒， 双酶切鉴定（见图2）并测定质粒内的HSV2病毒gE糖蛋白基因序列，DNA序列分析证实，重组质粒含有合成的HSV2病毒gE糖蛋白基因片段，序列完全正确：
GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCC ACT GCCACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGC GGA GAGGAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTG CTG TGGGCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCC CTG TGGCCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCT CCC GAACCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTG TAT AGTGAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATC TAC GGCGCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCC GAC GAGGCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCT ACT CCAACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGA CGC TCAGCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACC CAC CCACGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAG ACC CTGGAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATC CAC GCTATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTT GAT GTGCCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCT CAG CTGCCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCC TAC CGACTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGG TGT TTCGCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACA GTG AACCTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTG GTC TATGTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCA CAG TACAGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTG GAA CCAACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCC CTG CGTCAC CAC CAC CAC CAC CAT TGA T
构建的重组质粒表达HSV2的病毒gE糖蛋白片段（394个氨基酸），在其N端融合了载体上的19个氨基酸，在其C端融合了载体上的6个氨基酸，全长419个氨基酸,其氨基酸序列如下：
N端：Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr GlyVal His Ser
C端：His His His His His His
4．重组质粒在CHO细胞中的表达鉴定：
CHO细胞在不含血清的细胞表达培养基在锥形瓶中震荡培养，37℃，5% CO2。转染前一天，以适当的浓度将CHO细胞接种于不含血清的细胞表达培养基中，震荡悬浮培养，37℃，5% CO2。用DNA和PEI混合物转染重组质粒。转染后第五天收集细胞上清，检测蛋白表达水平，第六天收集所有上清用于纯化重组蛋白。
表达HSV2病毒gE糖蛋白的纯化
根据表达HSV2病毒gE糖蛋白的氨基酸序列，分析其理化特性，确定适当的纯化方法。CHO细胞表达的HSV2 gE糖蛋白融合有载体上的His蛋白,因此我们决定采用亲和层析法，用His TrapTM FF crude column进行纯化。纯化获得了表达的gE糖蛋白，具体步骤如下：
材料和方法
1.主要试剂:His TrapTM FF crude column为GE Heathcare公司产品，其它试剂为国产或进口分析纯试剂。
2. 重组蛋白的纯化：收集细胞上清100ml，离心后将上清以1ml/min的速度加入His TrapTM FF crude column中。10倍体积平衡液（1×PBS，0.5mol/L NaCl，20mmol/Limidazole，pH7.4）洗脱杂蛋白，再加入1ml洗脱液（1×PBS，0.5mol/L NaCl，500mmol/Limidazole，pH7.4）洗脱目的蛋白，静置20分钟后洗脱，收集洗脱液，用透析液（1×PBS，0.5mol/L NaCl，pH7.4）透析过夜，即为纯化的目的蛋白，SDS-PAGE、Western Blot检测，并测定蛋白浓度。
结果
将从His TrapTM FF crude column上洗脱的蛋白进行SDS-PAGE、Western Blot分析，结果显示(分别见图3、4)，表达产物约55kDa，蛋白浓度为27μg/ml。获得了表达的gE糖蛋白。
纯化HSV2病毒的gE糖蛋白的免疫原性分析
以纯化获得的gE2重组蛋白为抗原，混合弗氏佐剂免疫小鼠，制备抗血清，间接ELISA法检测效价，评测获得的gE2重组蛋白的免疫原性。
材料和方法
1.雄性昆明小鼠，6-8周龄，购自中国军事医学科学院实验动物中心。
2.完全弗氏佐剂和不完全弗氏佐剂为Sigma公司产品。小鼠抗His单抗、羊抗小鼠IgG-HRP为金斯特公司产品，其它试剂为国产或进口分析纯试剂。
3.动物免疫方法及抗血清制备：10只雄性昆明小鼠随机分为2组，每组5只。实验组以纯化的重组gE2糖蛋白与弗氏佐剂等体积混合，充分乳化，分别于第0，2，4周，腹腔注射免疫小鼠，2 ug/只/次，0.1ml/只，对照组以PBS等体积混合弗氏佐剂，充分乳化，0.1ml/只。每次免疫后2周眼眶采血。血液37℃放置1小时后4℃过夜，4000rpm离心10分钟，取上清即得重组蛋白gE2抗血清。间接ELISA检测抗血清滴度。
4.ELISA试验: 用1×CBS将纯化的重组gE2糖蛋白稀释为1 ug/ml，包被酶联板，每孔100ul，4℃过夜。次日5%FBS封闭酶联板，每孔130ul, 室温2小时。将制备的抗血清用样本稀释液按1:500，1:2500，1:12500逐级稀释后，分别加至封闭后的酶联板孔内，每孔100ul，37℃反应1小时，用PBST洗5遍后，加1:5000稀释的羊抗小鼠IgG-HRP，每孔100ul，37℃反应30 分钟，PBST洗5遍，加底物TMB溶液每孔100 ul，37℃避光显色10 分钟，加50ul 1N盐酸终止反应，用酶联仪测定A450值。
结果
每次免疫后2周小鼠眼底静脉丛取血，制备抗血清，间接ELISA法检测血清血清中抗体滴度。结果显示，小鼠抗gE2血清平均滴度在免疫前、第一次免疫后、第二次免疫后、第三次免疫后的抗血清滴度分别为0.00±0.00、4.10±0.28、5.49±0.28、6.89±0.28（如图5），且PBS对照组抗gE2血清滴度始终为0.00。小鼠在第一次免疫后即产生较高水平的抗体，通过第二次、第三次免疫后，小鼠体内抗体水平仍缓慢升高，产生高滴度水平。使用SPSS13.0软件进行单因素方差分析及Q检验，P＜0.05，免疫前及三次免疫后小鼠抗血清滴度的提高均具有显著统计学意义。表达的重组蛋白gE2能刺激免疫***产生较好的体液免疫效果。
化学合成的HSV病毒gE蛋白胞外区基因片段序列表见附件文档：核苷酸或氨基酸序列表计算机可读载体。
Claims (3)
1.一种化学合成的HSV2病毒gE糖蛋白的胞外区基因片段，该基因片段编码含强抗原表位的HSV2病毒的gE糖蛋白胞外区片段，即第21位氨基酸至第414位 氨基酸，共394个氨基酸，化学合成的基因序列全长1282 bp，序列如下：
GAATTC GCC GCC ACC ATG GGC TGG AGC TGT ATT ATC CTG TTC CTG GTC GCC ACTGCC ACT GGA GTT CAT TCT GCC GCC CCT CGG ACA TCT TGG AAA AGG GTG ACC AGC GGAGAG GAT GTG GTC CTG CTG CCT GCA CCA GCT GAA AGG ACA CGA GCA CAC AAG CTG CTGTGG GCA GCT GAG CCT CTG GAC GCA TGC GGT CCA CTG CGA CCA TCT TGG GTG GCC CTGTGG CCA CCT AGG CGG GTC CTG GAG ACC GTG GTC GAT GCA GCC TGT ATG AGA GCT CCCGAA CCT CTG GCA ATC GCC TAC TCT CCA CCC TTC CCA GCA GGC GAT GAG GGA CTG TATAGT GAA CTG GCT TGG AGA GAC CGC GTG GCA GTG GTC AAC GAG TCC CTG GTC ATC TACGGC GCC CTG GAA ACA GAT AGC GGA CTG TAT ACT CTG TCA GTG GTC GGA CTG TCC GACGAG GCA CGA CAG GTG GCT TCC GTG GTC CTG GTG GTC GAA CCA GCA CCA GTG CCT ACTCCA ACC CCA GAC GAT TAC GAC GAG GAA GAC GAT GCT GGC GTG ACT AAC GCA AGA CGCTCA GCC TTC CCT CCA CAG CCA CCT CCA AGG AGG CCC CCT GTG GCA CCA CCA ACC CACCCA CGC GTC ATC CCA GAG GTG TCC CAC GTC CGA GGA GTG ACT GTC CAT ATG GAG ACCCTG GAA GCT ATT CTG TTC GCA CCT GGG GAA ACA TTT GGT ACT AAT GTG AGT ATC CACGCT ATT GCA CAT GAC GAT GGA CCC TAT GCT ATG GAC GTG GTC TGG ATG CGA TTT GATGTG CCT TCC AGC TGC GCC GAC ATG AGG ATC TAC GAG GCT TGT CTG TAT CAT CCT CAGCTG CCA GAA TGC CTG TCC CCT GCC GAT GCT CCA TGT GCC GTG TCT AGT TGG GCC TACCGA CTG GCT GTG CGT AGC TAT GCT GGC TGC TCT AGG ACC ACA CCT CCA CCC CGG TGTTTC GCA GAG GCC AGA ATG GAA CCT GTG CCA GGA CTG GCT TGG CTG GCA AGT ACA GTGAAC CTG GAG TTT CAG CAC GCA TCA CCA CAG CAT GCC GGG CTG TAC CTG TGC GTG GTCTAT GTG GAC GAT CAC ATC CAT GCC TGG GGT CAT ATG ACC ATT AGC ACA GCT GCA CAGTAC AGG AAT GCT GTG GTC GAG CAG CAC CTG CCA CAG CGT CAG CCA GAG CCT GTG GAACCA ACC CGC CCT CAT GTC AGA GCA CCC CAC CCC GCC CCT TCA GCC AGA GGA CCC CTGCGT CAC CAC CAC CAC CAC CAT TGA TAAGCTT。
2.一种方法，包括采用基因工程技术表达如权利要求1所述的化学合成的HSV2病毒gE糖蛋白的胞外区基因片段，纯化表达的蛋白片段，具体步骤如下：
表达HSV2病毒的gE糖蛋白胞外区基因片段重组质粒的构建:
用EcoR I和Hind III双酶切权利要求1所述的化学合成的基因片段和质粒pMCE5，电泳回收后，用T4 DNA 连接酶连接，使该基因片段***到载体pMCE5内的EcoR I和Hind III位点之间，与载体上的起始密码子翻译框架一致，表达一个融合蛋白，该融合蛋白N端含有19个氨基酸的信号肽，C端包含HSV2病毒的gE糖蛋白内的第21位氨基酸至第414位氨基酸及6个His氨基酸标签；
表达的融合蛋白分泌到细胞外时，其N端的19个氨基酸信号肽被切除，最终分泌表达蛋白的氨基酸序列如下：
Ala Ala Pro Arg Thr Ser Trp Lys Arg Val Thr Ser Gly Glu Asp Val Val LeuLeu Pro Ala Pro Ala Glu Arg Thr Arg Ala His Lys Leu Leu Trp Ala Ala Glu ProLeu Asp Ala Cys Gly Pro Leu Arg Pro Ser Trp Val Ala Leu Trp Pro Pro Arg ArgVal Leu Glu Thr Val Val Asp Ala Ala Cys Met Arg Ala Pro Glu Pro Leu Ala IleAla Tyr Ser Pro Pro Phe Pro Ala Gly Asp Glu Gly Leu Tyr Ser Glu Leu Ala TrpArg Asp Arg Val Ala Val Val Asn Glu Ser Leu Val Ile Tyr Gly Ala Leu Glu ThrAsp Ser Gly Leu Tyr Thr Leu Ser Val Val Gly Leu Ser Asp Glu Ala Arg Gln ValAla Ser Val Val Leu Val Val Glu Pro Ala Pro Val Pro Thr Pro Thr Pro Asp AspTyr Asp Glu Glu Asp Asp Ala Gly Val Thr Asn Ala Arg Arg Ser Ala Phe Pro ProGln Pro Pro Pro Arg Arg Pro Pro Val Ala Pro Pro Thr His Pro Arg Val Ile ProGlu Val Ser His Val Arg Gly Val Thr Val His Met Glu Thr Leu Glu Ala Ile LeuPhe Ala Pro Gly Glu Thr Phe Gly Thr Asn Val Ser Ile His Ala Ile Ala His AspAsp Gly Pro Tyr Ala Met Asp Val Val Trp Met Arg Phe Asp Val Pro Ser Ser CysAla Asp Met Arg Ile Tyr Glu Ala Cys Leu Tyr His Pro Gln Leu Pro Glu Cys LeuSer Pro Ala Asp Ala Pro Cys Ala Val Ser Ser Trp Ala Tyr Arg Leu Ala Val ArgSer Tyr Ala Gly Cys Ser Arg Thr Thr Pro Pro Pro Arg Cys Phe Ala Glu Ala ArgMet Glu Pro Val Pro Gly Leu Ala Trp Leu Ala Ser Thr Val Asn Leu Glu Phe GlnHis Ala Ser Pro Gln His Ala Gly Leu Tyr Leu Cys Val Val Tyr Val Asp Asp HisIle His Ala Trp Gly His Met Thr Ile Ser Thr Ala Ala Gln Tyr Arg Asn Ala ValVal Glu Gln His Leu Pro Gln Arg Gln Pro Glu Pro Val Glu Pro Thr Arg Pro HisVal Arg Ala Pro His Pro Ala Pro Ser Ala Arg Gly Pro Leu Arg His His His HisHis His
重组质粒的筛选与鉴定：
将重组质粒转化大肠杆菌DH5α，涂布含100μg／ml氨苄青霉素的LB平板，置37℃ 过夜；
次日随机挑取转化菌落、1个含质粒pMCE5的对照菌转化菌，1个含质粒pUC57-gE2阳性对照菌，菌液PCR扩增HSV2病毒gE糖蛋白胞外区基因片段,含HSV2病毒gE糖蛋白胞外区基因片段的阳性重组质粒，应扩增出长1282bp 的基因片段；在提取阳性单菌落质粒，进行双酶切鉴定及测序；
重组质粒在CHO细胞中的表达鉴定：
CHO细胞在不含血清的细胞表达培养基在锥形瓶中震荡培养，37℃，5% CO2；
转染前一天，以适当的浓度将CHO细胞接种于不含血清的细胞表达培养基中，震荡悬浮培养，37℃，5% CO2；用DNA和PEI混合物转染重组质粒；转染后第五天收集细胞上清，检测蛋白表达水平，第六天收集所有上清用于纯化重组蛋白；
重组蛋白的纯化、鉴定和浓度检测：
收集细胞上清100ml，离心后将上清以1ml/min的速度加入His TrapTM FF crudecolumn中；10倍体积平衡液洗脱杂蛋白，平衡液为1×PBS，0.5mol/L NaCl，20mmol/Limidazole，pH7.4；再加入1ml洗脱液洗脱目的蛋白，洗脱液为1×PBS，0.5mol/L NaCl，500mmol/L imidazole，pH7.4；静置20分钟后洗脱，收集洗脱液，用透析液透析过夜，透析液为1×PBS，0.5mol/L NaCl，pH7.4，洗脱的蛋白即为纯化的目的蛋白，SDS-PAGE、WesternBlot检测，并测定蛋白浓度；
纯化获得的gE2蛋白分子量为55kD。
3.权利要求1所述的化学合成的HSV2病毒gE糖蛋白的胞外区基因片段序列，用于HSV2病毒亚单位疫苗的研制的用途。
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