CN116444628A - 一种狂犬病病毒的g蛋白及应用 - Google Patents
一种狂犬病病毒的g蛋白及应用 Download PDFInfo
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Abstract
本发明属于细胞重组与蛋白表达技术领域，具体涉及一种表达狂犬病病毒蛋白的构建方法和应用，所述一种表达狂犬病病毒蛋白为狂犬病病毒的重组G蛋白，所述重组G蛋白的基因序列为SEQ ID NO.1所示。本发明还成功建立了表达编码改造G蛋白的CHO悬浮细胞系，所述细胞系为CHO‑TEGH悬浮细胞系，其保藏信息如下，命名：中国仓鼠卵巢细胞CHO‑GH8；保藏单位：中国典型培养物保藏中心；保藏日期：2023年4月23日；保藏地址：武汉武汉大学保藏中心；保藏编号：CCTCCNO：C202389；该细胞系可稳定、高效表达具有优秀免疫原性的RABVG蛋白，具有应用于狂犬病新型亚单位疫苗、抗体诊断试剂的潜力。
Description
技术领域
本发明属于细胞重组与蛋白表达技术领域，具体涉及一种狂犬病病毒G蛋白的制备方法和应用。
背景技术
狂犬病(Rabies)是由狂犬病病毒(Rabies virus，RABV)引起的一种人兽共患的急性接触性传染病，临床的主要特征为神经兴奋和意识障碍，还伴有恐水、怕风、咽肌痉挛、继而造成局部或全身麻痹而死，可感染所有温血动物，感染发病后死亡率几乎达100％。狂犬病广泛分布于150多个国家和地区，全球每年约59,000人死于该病，中国是受狂犬病危害最严重的国家之一，仅次于印度。世界卫生组织(WHO)提出了“2030年清除犬传人间狂犬病”计划，我国一直积极响应该计划。几乎所有人间狂犬病均由犬传播，而我国犬只数量巨大，其中大量散养、流浪犬的存在也加大了人间狂犬病流行的风险，使我国狂犬病的清除工作仍面临巨大的挑战。狂犬病能完全通过疫苗接种来预防，目前针对狂犬病尚无特效疗法。开发安全、有效的人用和兽用狂犬病疫苗是当前国内外狂犬病的研究重点。此外，市场极度缺乏狂犬病抗体快速检测产品，可快速筛查人和动物RABV抗体，在临床和现地配合狂犬病疫苗的市场应用。
狂犬病毒属于弹状病毒科(Rhaboviridae)狂犬病病毒属(Lyssaavirus)，其基因组RNA严格保守有序地编码5种结构蛋白：核蛋白(N)，磷蛋白(P)，基质蛋白(M)，囊膜糖蛋白(G)和大聚合酶蛋白(L)。G蛋白存在于RABV病毒颗粒表面，G蛋白以同源三聚体的形式位于病毒囊膜表面，形成纤突，单体G蛋白由524个氨基酸组成，蛋白分子量约为62kD。G蛋白能激发机体产生细胞免疫，诱导产生中和抗体。G蛋白可以糖基化，在其表达及分泌过程中，糖基化修饰对其稳定性、抗原性及生物活性有着至关重要的作用。此外，G蛋白可介导细胞融合、受体结合以及参与病毒的致病等。G蛋白仍然是目前狂犬病疫苗、抗病毒药物和抗体诊断试剂研发的重要靶标蛋白。
中国仓鼠卵巢细胞(CHO)可高效容纳、扩增和表达外源蛋白。CHO不仅可在细胞内高效表达外源蛋白，还具有高效分泌外源蛋白至胞外的优势。CHO细胞表达外源蛋白的免疫原性等生物活性与天然蛋白相似，能够充分展示抗原表位，有效减少非特异性结合。目前，CHO表达系统成熟、可靠，下游纯化工艺简单，在研究基因表达及功能方面备受青睐。组织型纤溶酶原激活因子信号肽基因(Tissue plasminogen activator signal sequence,tPA)可提高异源蛋白的胞内转录、表达，能够高效引导蛋白的的胞外分泌，进而提高体液免疫水平。噬菌体T4纤维蛋白的C端结构域(T4-foldon domain)是纤维蛋白三聚体结构形成的必要条件，可作为人工三聚体结构域，应用于外源蛋白三聚体的重组表达。因此，为了高效表达、纯化胞外分泌的RABV G蛋白三聚体，本发明以CHO-K1悬浮表达系统为基础，构建了融合表达tPA信号肽、删除信号肽的哺乳动物密码子偏噬性密码子优化ERA G基因胞外区、T4foldon结构域和6×His标签蛋白的RABV G蛋白真核表达悬浮细胞系，以期获得高水平稳定表达、易纯化具有优秀血清特异性和免疫原性的G蛋白。通过免疫BALB/c小鼠初步评价其免疫原性，为狂犬病新型亚单位疫苗、抗体诊断试剂的研发奠定良好的基础。
发明内容
为了提高狂犬病病毒G蛋白的表达水平，本发明在优化G蛋白基因的基础上构建了表达重组G蛋白的CHO表达系统，从而完成了本发明。
第一方面，本发明提供一种狂犬病病毒的重组G蛋白，所述重组G蛋白的基因TEGH，其序列为SEQ ID NO.1所示。
第二方面，本发明提供一种含有第一方面重组G蛋白的狂犬病亚单位疫苗，所述疫苗可以进一步含有疫苗佐剂。
第三方面，本发明提供一种第一方面所述的重组G蛋白在制备狂犬病亚单位疫苗中的应用。
进一步，所述亚单位疫苗是指含有G蛋白的疫苗或含有G蛋白的核酸的疫苗。
第四方面，本发明提供一种第一方面所述的重组G蛋白在制备抗体诊断试剂中的应用。
第五方面，本发明提供重组G蛋白的制备方法，所述方法包括如下步骤：
S1选择ERAG蛋白胞外区基因，在其N末端用tPA信号肽基因替换G基因的信号肽基因；
S2在S1的基础上，在其C末端加入T4 foldon结构域和6×His基因序列。
S3通过编码改造得到重组G蛋白的基因TEGH，基因序列为SEQ ID NO.1所示，定向克隆至表达载体。
第六方面，本发明提供一种表达第一方面所述的重组G蛋白真核表达载体，所述含有重组G蛋白的真核表达载体pcDNA3.1-TEGH基因序列如SEQ ID NO.3。
第七方面，本发明提供一种表达第一方面的重组G蛋白的细胞系，所述细胞系为CHO-TEGH悬浮细胞系，其保藏信息如下，命名：中国仓鼠卵巢细胞CHO-GH8；保藏单位：中国典型培养物保藏中心；保藏日期：2023年4月23日；保藏地址：武汉武汉大学保藏中心；保藏编号：CCTCC NO：C202389。
附图说明
图1.细胞系CHO-TEGH的构建与鉴定(A为真核表达载体pcDNA3.1-TEGH的构建；B为CHO-TEGH的间接免疫荧光鉴定；C为CHO-TEGH上清纯化蛋白的SDS-PAGE鉴定；D为CHO-TEGH上清纯化蛋白的Western-blot鉴定)。
图2：悬浮细胞系CHO-GH8表达TEGH的传代稳定性分析(A为不同代次细胞培养上清蛋白的SDS-PAGE分析；B为不同代次细胞培养上清纯化蛋白Western-blot分析)。其中，中国仓鼠卵巢细胞CHO-GH8；保藏单位：中国典型培养物保藏中心；保藏日期：2023年4月23日；保藏地址：武汉武汉大学保藏中心；保藏编号：CCTCC NO：C202389。
图3：TEGH蛋白在BALB/c小鼠上的免疫原性分析。
具体实施方式
下面对本发明的具体实施方式作进一步说明。在此需要说明的是，对于这些实施方式的说明用于帮助理解本发明，但并不构成对本发明的限定。此外，下面所描述的本发明各个实施方式中所涉及的技术特征只要彼此之间未构成冲突就可以相互组合。
下述实施例中的实验方法，如无特殊说明，均为常规方法，下述实施例中所用的试验材料，如无特殊说明，均为可通过常规的商业途径购买得到。
实施例1稳定表达TEGH蛋白CHO单克隆细胞系的构建
1.实验材料
中国仓鼠卵巢细胞CHO-K1(ATCC，CCL-61)由本实验室保存。F12K培养液、Opti-MEM培养液、胎牛血清均购自Gibco公司；X-tremeGENE HP DNA Transfection Reagent转染试剂购自ROCHE公司；HighAffinityNi-Charged Resin FF购自GenScript公司；G418、咪唑购自Amresco公司；考马斯亮蓝染色液、脱色液购自Biosharp公司；Tween-20、FITC标记山羊抗小鼠IgG购自Sigma公司；纯化镍柱购自GenScript公司；抗RABV鼠血清由本实验室用ERA株免疫小鼠制备；青链霉素混合液、PBS购自索莱宝公司，红外荧光标记的驴抗小鼠IgG二抗购自Abcam。倒置荧光显微镜购自Zeiss公司；蛋白质凝胶电泳系统购自Bio-Rad公司；Odyssey红外荧光扫描成像仪购自Li-cor公司。
2.真核表达载体pcDNA3.1-TEGH的构建
选择狂犬病病毒rERA株糖蛋白(G)基因胞外区核苷酸序列(GenBank:GQ406342.1)作为靶标抗原基因。为了高效表达分泌型G蛋白，将G基因进行人工编码改造为TEGH，包括：ERAG蛋白胞外区基因进行仓鼠密码子偏噬性密码子优化，在其N末端用tPA信号肽基因替换G基因的信号肽基因，在C末端加入T4 foldon结构域和6×His基因序列。通过人工合成编码改造的基因TEGH，定向克隆至真核表达载体pDNA3.1(+)(InvitrogenTM，V79020)，构建真核表达载体pcDNA3.1-TEGH(见图1A)。
3.TEGH蛋白稳定表达细胞系的建立
CHO-K1细胞铺六孔培养板过夜，使细胞贴壁率达到80％，按照X-tremeGENE HPDNA Transfection Reagent转染试剂的操作说明将pcDNA3.1-TEGH质粒转染细胞作为筛选细胞组，同时设置未转染pcDNA3.1-TEGH质粒的CHO-K1细胞为对照细胞组。转染后48h将细胞培养液更换成选择性培养液(F12k含10％血清、1％双抗+1000ug/mL G418)进行加压筛选，3天更换相同配方的筛选培养基。当对照细胞组细胞全部死亡时，用胰酶消化筛选细胞组剩余所有细胞，通过有限稀释法将存活的细胞以每孔1个细胞接种于96孔板中。待96孔板上的单克隆细胞长至汇合度约50％左右时，消化细胞，转移至两块同样的96孔板中，其中一块用于筛选后扩大培养，另一块待48h后进行IFA鉴定。单克隆细胞株经过免疫荧光初步鉴定后为阳性后，挑出扩大培养，收集上清，采用Western-blot方法进一步检测蛋白表达情况，筛选高表达的单克隆细胞株。
4.间接免疫荧光(IFA)
用预冷的3％多聚甲醛固定细胞30min，PBS洗3遍。以1：100倍稀释的抗RABV鼠血清为一抗，孵育1h，PBST(0.05％Tween20)洗3遍；以1：100倍稀释的绿色荧光素(FITC)标记的山羊抗鼠IgG为二抗，孵育染色1h，PBST洗3遍；在荧光显微镜下观察结果(见图1B)。
5.蛋白的纯化与鉴定
将IFA显示特异性荧光染色强度较强的单克隆细胞筛选扩大培养，收集细胞上清，借助His标签，通过亲和层析法用镍柱进行蛋白纯化，分别用含80mM、200mM、350mM、500mM咪唑的Elution buffer洗脱，收集目的蛋白，纯化后透析除去蛋白溶液中的咪唑，获得TEGH蛋白。通过UV法测定蛋白浓度，并分别取100uL进行SDS-PAGE和Western-blot鉴定，检测蛋白表达情况。高表达量的单克隆细胞株扩大培养至细胞瓶中，命名为F6代次CHO-TEGH，冻存于液氮中。
为鉴定CHO-TEGH的RABV G蛋白表达情况，实验结果显示，IFA染色后的CHO-TEGH所有细胞均有绿色荧光，呈现针对RABV G蛋白抗体的阳性结果(图1B)，表明CHO-TEGH的RABVG蛋白可有效表达，并具有RABV相应的生物学活性。
为鉴定蛋白TEGH纯化效果，将收集到的不同条件下纯化的目的蛋白进行SDS-PAGE电泳，考马斯亮蓝染色结果显示用含200mM、350mM、500mM咪唑的Elutionbuffer洗脱后纯化均在约67kDa处出现目的蛋白条带，与预期大小相符，且条带粗细无明显差异(见图1C)，表明200mM咪唑浓度可有效纯化TEGH蛋白。
为进一步鉴定纯化TEGH蛋白的特异性，以抗RABV鼠血清作为一抗，红外荧光标记的驴抗小鼠IgG作为二抗进行Western-blot分析，结果显示抗体反应特异性条带与SDS-PAGE的一致(见图1D)，表明TEGHG蛋白在单克隆细胞系CHO-TEGH中正确表达，并具有良好的特异性抗原抗体反应。
实施例2悬浮细胞系CHO-GH8表达TEGH蛋白及鉴定
1.CHO-TEGH细胞悬浮驯化
取对数生长期的F6代次CHO-TEGH贴壁细胞，每隔2-4天采用逐步降血清法将含10％FBS的F12K培养基逐步替换为8％、5％、3％、2％、1％、0.5％FBS的F12K培养基进行传代至F13代。将F13代CHO-TEGH以5×105cells/mL的密度接种到含0.5％FBS的100mLF12K培养基中，在250mL三角细胞摇瓶中按37℃、5％CO2、100rpm的条件进行去血清和悬浮驯化传代至F18代，获得无血清悬浮细胞系F1代次CHO-GH8，冻存于液氮中。
2.悬浮细胞系GH8的鉴定及遗传稳定性分析
将CHO-GH8(细胞系：中国仓鼠卵巢细胞CHO-GH8；保藏单位：中国典型培养物保藏中心；保藏日期：2023年4月23日；保藏地址：武汉武汉大学保藏中心；保藏编号：CCTCC NO：C202389。)按37℃、5％CO2、100rpm、无血清培养基的条件培养4天，收获悬浮细胞培养上清，并按同样的条件传代，连续传代培养20代，收取每代次悬浮细胞培养上清进行SDS-PAGE鉴定。随后将各代次上清过滤去除细胞碎片后，按照1.5所述方法进行蛋白纯化。通过UV法测定蛋白浓度并分装保存于-80℃冰箱。取不同代次的100uL纯化蛋白进行Western-blot鉴定，检测不同代次蛋白表达情况，以分析细胞系的TEGH表达和连续传代稳定性。
实验结果显示，考马斯亮蓝染色结果显示细胞系F2至F20代次细胞上清均可在约67kDa处出现目的蛋白条带，与预期大小相符，且条带粗细无明显差异，表明CHO-GH8细胞系表达TEGH蛋白具有良好的传代稳定性(见图2A)。各代次上清蛋白纯化后进行Western-blot，结果显示F2至F20各代次抗体反应特异性条带与SDS-PAGE的一致，表明TEGHG蛋白在CHO-GH8的连续传代中可稳定、正确表达，并具有良好的特异性抗原抗体反应(见图2B)。
实施例3TEGH蛋白免疫原性评价
取F5代次CHO-GH8(细胞系：中国仓鼠卵巢细胞CHO-GH8；保藏单位：中国典型培养物保藏中心；保藏日期：2023年4月23日；保藏地址：武汉武汉大学保藏中心；保藏编号：CCTCC NO：C202389。)培养4天的上清培养液2L，纯化获得浓度约为2mg/mL的TEGH蛋白1mL。为了评估重组蛋白TEGH蛋白的免疫原性，将16只6周龄的BALB/c小鼠，随机分为蛋白免疫组和PBS对照组，8只/组。TEGH混入10％的MontanideTMGEL 02(SEPPIC)佐剂，按5ug/100uL/只的剂量肌肉接种8只小鼠作为蛋白免疫组；同时，用PBS按100uL/只的剂量接种8只小鼠作为PBS对照组。初次免疫后21天按相同剂量、途径加强免疫。所有小鼠分别于初次免疫第0、21、35天经眶下静脉丛采血，分离血清。血清经56℃水浴灭活30min后进行FAVN法进行RABV中和抗体检测(见图3)。
实验结果显示所有8只免疫小鼠在初免后21天均可产生RABV中和抗体，抗体效价在1.97-10.26IU/mL之间，平均滴度为6.21IU/mL；加强免疫后中和抗体水平显著增高，抗体效价在40.50-70.15IU/mL之间，平均滴度为56.42IU/mL，表明CHO-GH8细胞系表达的TEGH蛋白在BALB/c小鼠上具有优秀的免疫原性。
本发明成功建立了CHO平台稳定表达RABV G蛋白的单克隆悬浮细胞系CHO-GH8(细胞系：中国仓鼠卵巢细胞CHO-GH8；保藏单位：中国典型培养物保藏中心；保藏日期：2023年4月23日；保藏地址：武汉武汉大学保藏中心；保藏编号：CCTCC NO：C202389。)，其细胞培养上清可高效表达、纯化TEGH蛋白，并具有传代稳定性。纯化的TEGH蛋白具有优秀的抗原抗体反应特性和小鼠体内免疫原性，具有应用于狂犬病亚单位疫苗和病毒抗原、抗体相关诊断试剂的潜力。
Claims (8)
1.一种狂犬病病毒的重组G蛋白，所述重组G蛋白的TEGH基因，其基因序列为SEQ IDNO.1所示。
2.一种含有权利要求1所述的重组G蛋白的狂犬病亚单位疫苗，所述疫苗还可以含有重组G蛋白的疫苗佐剂。
3.一种如权利要求1所述的重组G蛋白在制备狂犬病亚单位疫苗中的应用。
4.如权利要求3所述重组G蛋白在制备狂犬病亚单位疫苗中的应用，其特征在于，所述亚单位疫苗是指含有G蛋白的疫苗或含有G蛋白的核酸的疫苗。
5.一种如权利要求1所述的重组G蛋白在制备抗体诊断试剂中的应用。
6.一种重组G蛋白的制备方法，所述方法包括如下步骤：
S1选择ERAG蛋白胞外区基因，在其N末端用tPA信号肽基因替换G基因的信号肽基因；
S2在S1的基础上，在其C末端加入T4foldon结构域和6×His基因序列；
S3通过编码改造得到重组G蛋白的基因TEGH，基因序列为SEQ ID NO.1所示，定向克隆至表达载体。
7.一种表达如权利要求1所述的重组G蛋白的真核表达载体，所述含有重组G蛋白的真核表达载体pcDNA3.1-TEGH基因序列如SEQ ID NO.3。
8.一种表达如权利要求1所述的重组G蛋白的细胞系，所述细胞系为CHO-TEGH悬浮细胞系，命名：中国仓鼠卵巢细胞CHO-GH8；保藏单位：中国典型培养物保藏中心；保藏日期：2023年4月23日；保藏地址：武汉武汉大学保藏中心；保藏编号：CCTCCNO：C202389。
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