CN107973850A - 重组猪瘟病毒e2蛋白猪源化单克隆抗体及其制备方法和应用 - Google Patents
重组猪瘟病毒e2蛋白猪源化单克隆抗体及其制备方法和应用 Download PDFInfo
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- CN107973850A CN107973850A CN201711330540.6A CN201711330540A CN107973850A CN 107973850 A CN107973850 A CN 107973850A CN 201711330540 A CN201711330540 A CN 201711330540A CN 107973850 A CN107973850 A CN 107973850A
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Abstract
本发明公开了重组猪瘟病毒E2蛋白猪源化单克隆抗体及其制备方法和应用。本发明首先公开了重组猪瘟病毒E2蛋白猪源化单克隆抗体，其重链的氨基酸序列为SEQ ID No.1所示，轻链的氨基酸序列为SEQ ID No.2所示。本发明还公开了稳定表达重组猪瘟病毒E2蛋白猪源化单克隆抗体的悬浮HEK293细胞系。本发明通过将重组猪瘟病毒E2蛋白猪源化单克隆抗体的重链和轻链编码基因克隆至真核表达载体，利用悬浮HEK293细胞实现重组猪瘟病毒E2蛋白猪源化单克隆抗体的稳定和高效表达。本发明所述重组猪瘟病毒E2蛋白猪源化单克隆抗体具有良好的反应原性和中和活性，在开发新型猪瘟病毒诊断和治疗制剂中具有重要应用价值。
Description
技术领域
本发明涉及一种猪瘟病毒E2蛋白的猪源化单克隆抗体，进一步涉及所述猪源化单克隆抗体的制备方法，本发明还涉及猪瘟病毒E2蛋白的猪源化单克隆抗体在制备猪瘟病毒诊断试剂或抗病毒制剂中的应用，属于重组猪瘟病毒E2蛋白猪源化单克隆抗体的制备领域。
背景技术
猪瘟(classical swine fever，CSF)是严重危害养猪业的一种烈性传染病，常造成巨大的经济损失，该病被世界动物卫生组织(OIE)列为必须申报的动物疫病。CSF的病原是猪瘟病毒(classical swine fever virus，CSFV)，CSFV是有囊膜的单股正链RNA病毒，基因组长约12.3kb，是黄病毒科瘟病毒属的成员。该基因组仅含有一个大的开放阅读框，其编码一个由3,898个氨基酸组成的多聚蛋白，该多聚蛋白被裂解为4种结构蛋白(C和Erns、E1和E2)及8种非结构蛋白(Npro、P7、NS2、NS3、NS4A、NS4B、NS5A和NS5B)。
E2蛋白在病毒生命周期中发挥重要的作用，影响病毒的吸附、组织嗜性和病毒毒力。此外，E2是CSFV主要的保护性抗原，诱导机体产生中和抗体以抵抗病毒的感染(Beer M,Goller KV,Staubach C,Blome S.2015.Genetic variability and distribution ofClassical swine fever virus.Anim Health Res Rev 16:33–39.)。E2蛋白在氨基端有B、C、D、A四个抗原结构域，构成两个独立的抗原区，按B/C和A/D的顺序排列(氨基酸[aa]690–800和766–865)(Hulst MM,Westra DF,Wensvoort G,Moormann RJ.1993.GlycoproteinE1of hog cholera virus expressed in insect cells protects swine from hogcholera.J Virol 67:5435–5442；M,Lengsfeld T,Pauly T,Stark R,ThielHJ.1995.Classical swine fever virus:independent induction of protectiveimmunity by two structural glycoproteins.J Virol 69:6479–6486；Wensvoort G,Terpstra C,de Kluijver EP,Kragten C,Warnaar JC.1989.Antigenic differentiationof pestivirus strains with monoclonal antibodies against hog choleravirus.Vet Microbiol 21:9–20.)。
E2蛋白的抗原特性随着单克隆抗体的陆续鉴定而得到解析。在前期研究中，本发明人实验室制备了一株鼠源E2蛋白单克隆抗体HQ06，其可以特异性识别CSFV C株和石门株E2蛋白的线性表位，该单克隆抗体重链为IgG1型、轻链为к型(侯强,彭伍平,孙元,仇华吉.2008.猪瘟病毒E2蛋白主要抗原区编码基因的原核表达及其单克隆抗体的制备.中国兽医科学38:1–5.)。
由于在制备单克隆抗体的传统方法中，制备抗体的时间长，并且因杂交瘤细胞并不能长期保存，有可能造成抗体细胞活力下降、抗性丢失。为了克服产生单克隆抗体的杂交瘤细胞不稳定、抗性易丢失等问题，将HQ06抗体重链和轻链基因克隆至真核表达载体，通过真核表达的方式表达抗体，并将抗体猪源化，将为研究CSFV E2蛋白结构、功能以及开发新型的CSFV诊断和治疗制剂奠定基础。
发明内容
本发明所要解决的第一个技术问题是提供一种重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw，该单克隆抗体具有良好的反应原性和中和活性；
本发明所要解决的第二个技术问题是提供一种重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw的制备方法，该方法将rHQ06Sw的重链和轻链的编码基因克隆至真核表达载体，应用悬浮HEK293细胞实现了抗体的稳定和高效表达；
本发明所要解决的第三个技术问题是提供稳定表达所述重组猪瘟病毒E2蛋白猪源化单克隆抗体的悬浮HEK293细胞系以及所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw在制备CSFV诊断或治疗制剂中的应用。
为解决上述技术问题，本发明所采取的技术方案是：
本发明首先公开了一种重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw，其重链(HC)的氨基酸序列为SEQ ID No.1所示，轻链(LC)的氨基酸序列为SEQ ID No.2所示。
本发明进一步公开了所述重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw的重链和轻链的编码基因。
本发明通过提取HQ06杂交瘤细胞和猪淋巴细胞的总RNA，经反转录制备cDNA，然后分别以HQ06抗体可变区基因和猪源抗体恒定区基因为模板，利用融合PCR方法分别扩增获得rHQ06Sw重链(HC)和轻链(LC)全长序列。其中，rHQ06Sw重链的编码基因的核苷酸序列为SEQ ID No.3所示，轻链的编码基因的核苷酸序列为SEQ ID No.4所示。
本发明进一步公开了一种所述重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw的制备方法，包括以下步骤：将重组猪瘟病毒E2蛋白猪源化单克隆抗体的重链和轻链的编码基因(分别为SEQ ID No.3、SEQ ID No.4所示)分别克隆至真核表达载体，共转染宿主细胞，构建共表达抗体重链和轻链的细胞系；收获所表达的重组抗体，纯化，即得。其中，所述宿主细胞选自HEK293T细胞(人胚肾细胞)、CHO细胞(中国仓鼠卵巢细胞)或悬浮HEK293细胞中的任意一种，优选为悬浮HEK293细胞。所述真核表达载体为慢病毒载体；优选的，所述慢病毒载体为pFUGW。
进一步优选的，所述重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw的制备方法，包括以下步骤：(1)将重组猪瘟病毒E2蛋白猪源化单克隆抗体的重链和轻链的编码基因分别与慢病毒载体pFUGW可操作的连接，分别得到含有重链编码基因的重组质粒pFUGW-HC和含有轻链编码基因的重组质粒pFUGW-LC；(2)将重组质粒pFUGW-HC以及辅助质粒共转染细胞，得到包装重组质粒pFUGW-HC的慢病毒；将重组质粒pFUGW-LC以及辅助质粒共转染细胞，得到包装重组质粒pFUGW-LC的慢病毒；(3)将包装重组质粒pFUGW-HC的慢病毒和包装重组质粒pFUGW-LC的慢病毒共转导细胞，得到共表达抗体重链和轻链的细胞系；应用有限稀释法获得单个细胞克隆，检测这些克隆细胞的稳定性和表达抗体的能力，进一步扩大培养，收获所表达的重组抗体，纯化，即得。
其中，步骤(1)所述重链的编码基因的核苷酸序列为SEQ ID No.3所示，所述轻链的编码基因的核苷酸序列为SEQ ID No.4所示。步骤(2)所述辅助质粒包括pMD2.G和psPAX2；步骤(2)所述细胞为HEK293T细胞。步骤(3)所述细胞选自HEK293T细胞、CHO细胞或悬浮HEK293细胞中的任意一种，优选为悬浮HEK293细胞。
本发明通过将rHQ06Sw重链和轻链基因分别克隆至质粒载体pFUGW，成功构建重组质粒pFUGW-HC和pFUGW-LC。本发明进一步分别将重组质粒pFUGW-HC和pFUGW-LC以及辅助质粒pMD2.G和psPAX2共转染至HEK293T细胞中，制备包装pFUGW-HC和pFUGW-LC质粒的慢病毒。将慢病毒分别共转导HEK293T、CHO和悬浮HEK293细胞，构建共表达rHQ06Sw抗体重链和轻链的细胞系。
本发明应用制备型液相层析系统AKTA purifier分别对HEK293T、CHO和悬浮HEK293细胞表达的猪源rHQ06Sw抗体进行纯化，相比于传统纯化蛋白的方法更高效，可在短时间内，在大量流入样品的情况下，根据紫外峰值快速精准的收取目的蛋白。双抗体夹心ELISA检测抗体与兔抗猪IgG的反应结果表明，三种细胞表达的抗体均能与兔抗猪IgG反应，其中悬浮HEK293细胞表达的rHQ06Sw与兔抗猪IgG的反应性最好。因此，本发明选用悬浮HEK293细胞制备rHQ06Sw抗体。
本发明进一步公开了稳定表达所述重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw的悬浮HEK293细胞系。
本发明用悬浮HEK293细胞制备rHQ06Sw抗体，进一步利用有限稀释法获得单个细胞克隆，检测这些克隆细胞的活力和表达抗体的能力，结果表明4#细胞克隆表达的抗体与兔抗猪IgG的反应性最好。
本发明将稳定表达所述重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw的悬浮HEK293细胞的4#细胞克隆提交专利认可的机构进行保藏，其微生物保藏编号为：CGMCCNo.14721；分类命名为：重组猪瘟病毒E2蛋白猪源化单克隆抗体细胞系；保藏单位：中国微生物菌种保藏管理委员会普通微生物中心；保藏时间是2017年9月20日；保藏地址：北京市朝阳区北辰西路1号院3号。
将鼠源抗体重链和轻链的可变区嵌合到人源抗体的恒定区的报道有很多(Steplewski Z,Sun LK,Shearman CW,Ghrayeb J,Daddona P,KoprowskiH.1988.Biological activity of human-mouse IgG1,IgG2,IgG3,and IgG4chimericmonoclonal antibodies with antitumor specificity.Proc Natl Acad Sci U S A 85:4852–4856；Shaw DR,Khazaeli MB,Sun LK,Ghrayeb J,Daddona PE,McKinney S,LoBuglioAF.1987.Characterization of a mouse/human chimeric monoclonal antibody(17-1A)to a colon cancer tumor-associated antigen.J Immunol138:4534–4538.)，但鲜有将鼠源抗体嵌合到猪源抗体中的报道。在前期研究中，本发明人实验室制备了一株鼠源E2蛋白单克隆抗体HQ06，本发明将实验室已有的鼠源抗体可变区嵌合入猪源抗体的恒定区，使之变为猪源抗体。由于抗体源性的改变，有可能造成抗体蛋白糖基化与折叠的改变，蛋白大小也可能随之所变化。本发明应用非还原性SDS-PAGE的检测结果表明，rHQ06Sw抗体成功组装成为160kDa大小的蛋白；还原的SDS-PAGE检测显示，rHQ06Sw抗体重链为55kDa、轻链为25kDa，与抗体蛋白的分子量一致。经过质谱分析，此蛋白确实为完整的rHQ06Sw抗体蛋白。Western blotting分析表明，rHQ06Sw可以和抗猪IgG抗体反应。双抗体夹心ELISA验证结果也表明，rHQ06Sw与兔抗猪IgG有强烈的反应。以上结果证明，rHQ06Sw抗体成功组装，并且可被兔抗猪IgG识别，说明抗体已经成功猪源化。
本发明应用间接ELISA、Western blotting和IPMA验证了rHQ06Sw抗体和CSFV E2蛋白的反应性。间接ELISA结果表明，rHQ06Sw抗体与不同表达系统表达的E2蛋白有良好的反应性，并且呈剂量依赖性。IPMA实验进一步证明了rHQ06Sw可以识别CSFV E2蛋白，具有良好的反应原性。Western blotting结果同样表明，rHQ06Sw抗体能够识别不同系统表达的E2，并且能与内源性CSFV E2蛋白反应，进一步证明rHQ06Sw抗体的特异性。综上，rHQ06Sw抗体与CSFVE2蛋白有良好的反应性。
本发明应用SPR试验检测了rHQ06Sw抗体与CSFV E2蛋白的结合力。传感图显示，rHQ06Sw抗体可与CSFV E2蛋白相互作用，并且这种作用呈剂量依赖性，表明rHQ06Sw抗体可以特异性识别并结合CSFV E2蛋白。本发明应用1:1结合模型分析，ka值为9.839×104M-1·s-1，KD值为1.308×10-8M，表明rHQ06Sw与CSFV E2蛋白具有良好的反应性。以上结果证实，rHQ06Sw抗体与CSFV E2蛋白具有很高的亲和力。
为了检测rHQ06Sw抗体与瘟病毒属其他成员的交叉反应，本发明应用间接ELISA检测rHQ06Sw抗体与牛病毒性腹泻病毒(BVDV)E2蛋白的反应性。结果表明，BVDV E2蛋白与抗BVDV猪源阳性血清有良好的反应性，并且呈剂量依赖性，但不与rHQ06Sw抗体反应。结果表明，rHQ06Sw抗体与BVDV无交叉反应。
中和活性鉴定结果表明，rHQ06Sw抗体具有中和CSFV的活性，可以有效中和CSFV；这为抗体治疗性应用及开发新型CSFV治疗性药物奠定了基础。
本发明进一步公开了所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw在制备猪瘟病毒(CSFV)诊断制剂中的应用。
本发明还公开了所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw在制备猪瘟病毒治疗制剂中的应用。
本发明还公开了所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw在制备猪瘟病毒抗体检测试剂中的应用。
本发明还公开了稳定表达所述重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw的悬浮HEK293细胞系在制备猪瘟病毒诊断制剂、治疗制剂或者猪瘟病毒抗体检测试剂中的应用。
本发明进一步公开了一种用于猪瘟病毒抗体检测的竞争ELISA试剂盒，其中包括：HRP(辣根过氧化物酶)标记的本发明所述重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw。
本发明证实了rHQ06Sw抗体的反应原性和抑制病毒复制的能力，并且有较好的亲和力。因此，rHQ06Sw在CSFV检测方面具有很高的应用价值。
本发明技术方案与现有技术相比，具有以下有益效果：
本发明构建了针对猪瘟病毒E2蛋白的猪源化单克隆抗体rHQ06Sw，并应用悬浮HEK293细胞实现了稳定和高效的表达，通过收集培养细胞上清获得抗体，使抗体的制备更加便捷。本发明所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw具有良好的反应原性和中和活性，在研究CSFV E2蛋白结构、功能以及开发新型的CSFV诊断和治疗制剂中具有重要的应用价值。
附图说明
图1为pFUGW-HC和pFUGW-LC重组质粒构建；其中，A：PCR扩增出rHQ06Sw重链、轻链目的基因；1-7泳道为HC、LC基因；8泳道为阴性对照；B：pFUGW-HC和pFUGW-LC的酶切鉴定结果；1：pFUGW-HC；2：pFUGW-LC；3：pFUGW；
图2为rHQ06Sw猪源抗体细胞系的筛选；其中，A为HEK293T、CHO和悬浮HEK293细胞表达的rHQ06Sw抗体与兔抗猪IgG的反应性；B为不同的悬浮HEK293细胞克隆表达的rHQ06Sw抗体与兔抗猪IgG的反应性，1-20分别为1-20#细胞克隆；
图3为rHQ06Sw猪源抗体活性的检测；其中，A-B：用SDS-PAGE检测rHQ06Sw抗体的组装；C-D：rHQ06Sw与兔抗猪IgG的反应性；Purified rHQ06Sw为纯化的rHQ06Sw；
图4为rHQ06Sw抗体的反应原性检测；其中，A-B：间接ELISA；C：IPMA；D-E：Westernblotting；Positive serum为阳性血清；Negative serum为阴性血清；
图5为SPR检测rHQ06Sw抗体与猪瘟病毒E2蛋白的结合力；
图6为用间接ELISA检测HQ06Sw抗体与BVDV的交叉反应；
图7为中和试验检测rHQ06Sw抗体对CSFV的中和作用；其中，A：rHQ06Sw抗体2倍倍比稀释；B：CSFV10倍倍比稀释。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但是应理解所述实施例仅是范例性的，不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改或替换均落入本发明的保护范围。
实施例1重组猪瘟病毒E2蛋白猪源化单克隆抗体的制备
1、材料和方法
1.1细胞、病毒和质粒
人胚肾细胞(HEK293T)和猪肾细胞(PK-15)购自美国标准菌种收藏中心；中国仓鼠卵巢细胞(CHO)购于中国科学院细胞库；悬浮HEK293细胞由哈尔滨动物生物制品国家工程研究中心有限公司提供。CSFV石门株由本实验室保存。抗CSFV E2蛋白单克隆抗体WH303由英国国家兽医实验室Trevor W.Drew教授馈赠。BVDV猪源阳性血清由汉诺威兽医大学OIE/欧盟猪瘟参考实验室Paul Becher教授馈赠。
HEK293T、PK-15细胞用含有10％胎牛血清(FBS)(Sigma-Aldrich公司)的DMEM培养；CHO细胞用含有10％FBS的DME/-F12培养基(HyClone公司)进行培养；悬浮的HEK293细胞用293ProTM CD 293S无血清培养基(BasalMedia Technologies公司)进行培养。
慢病毒载体pFUGW及辅助质粒psPAX2、pMD2.G由Addgene提供。
1.2质粒的构建
提取HQ06杂交瘤细胞和猪淋巴细胞的总RNA，经反转录制备cDNA。应用可变区引物(mVH-F、mVH-R、mVL-F和mVL-R)和恒定区引物(pCH-F、pCH-R、pCL-F和pCL-R)分别扩增HQ06抗体可变区和猪源抗体恒定区的序列。以鉴定为阳性的克隆质粒为模板，利用mVH-F、pCH-R、mVL-F和pCL-R通过融合PCR的方法扩增rHQ06Sw重链和轻链全长序列。引物序列见表1。琼脂糖凝胶电泳鉴定核酸分子量正确后，将目的条带胶回收并在37℃条件下酶切4h，将其与同样酶切线性化的质粒载体pFUGW于16℃连接8h，制备重组质粒pFUGW-HC和pFUGW-LC。
表1特异性引物序列
1.3构建共表达rHQ06Sw抗体重链和轻链的细胞系
将HEK293T细胞铺于直径为10-cm的细胞培养皿中，12h后分别将质粒pFUGW-HC和pFUGW-LC以及辅助质粒pMD2.G和psPAX2共转染至HEK293T细胞中，转染后48h收集细胞培养上清，制备包装pFUGW-HC和pFUGW-LC质粒的慢病毒。将慢病毒分别以10倍感染复数(MOI)共转导HEK293T、CHO和悬浮HEK293细胞，培养6h后，弃掉上清液，加入新的培养基，置于37℃温箱中进行培养，72h之后进行传代。
1.4rHQ06Sw的表达和纯化
应用制备型液相层析系统AKTA purifier分别对HEK293T、CHO和悬浮HEK293细胞表达的rHQ06Sw抗体进行纯化。首先，应用pH为8.0的平衡缓冲液(100mM Tris-HCl、150MNaCl、1mM EDTA)对HiTrap Protein A HP预装柱(catalog no.17-0403-01；GEHealthcare)进行清洗；之后将上清以3mL/min的速度流穿过柱；最后，应用3M甘氨酸(pH2.7)以2mL/min的速度进行洗脱，并应用Tris-HCl(pH 9.0)中和。筛选出最优细胞系之后，分别应用12％的还原性十二烷基硫酸钠-聚丙烯酰胺电泳(SDS-PAGE)和8％的非还原性SDS-PAGE检测抗体组装，并应用免疫印迹方法分析rHQ06Sw的组装以及与兔抗猪IgG的反应性。
1.5双抗体夹心酶联免疫吸附试验(DS-ELISA)
将纯化的鼠抗猪IgG(catalog no.552554；BD Pharmingen)以0.2μg/孔包被ELISA反应板。应用PBST洗涤后，加入含有5％FBS的5％脱脂乳对其进行封闭。将纯化的rHQ06Sw 2倍系列稀释后加入到ELISA反应板中，于37℃作用1h后，弃掉抗体，应用PBST清洗3次。加入辣根过氧化物酶(HRP)标记的兔抗猪IgG(1:4 000)(catalog no.ab6777；Abcam)，于37℃继续孵育1h。应用PBST洗涤3次，加入TMB(catalog no.T0040；Sigma)作用15min，用2M H2SO4终止反应，应用酶标仪(catalog no.ELx808；BioTek)在450nm波长处检测吸光度。
1.6间接ELISA检测rHQ06Sw与CSFV E2蛋白的反应活性
分别应用巴斯德毕赤酵母表达的E2蛋白、BHK-21和CHO细胞表达的E2蛋白以400ng/孔包被ELISA反应板，同时将纯化的CSFV以104TCID50/孔包被于ELISA反应板中，应用5％脱脂乳于37℃封闭2h。将纯化的rHQ06Sw进行2倍系列稀释后加入其中，于37℃孵育1h。应用PBST洗涤3次，加入HRP标记的兔抗猪IgG(H&L)(1:4 000)，37℃孵育1h后，加入TMB底物进行显色，应用酶标仪在450nm波长处检测吸光度。
1.7免疫过氧化物酶单层细胞试验(IPMA)
将PK-15细胞铺于96孔细胞培养板，接种CSFV(MOI＝0.1)，于37℃5％CO2温箱中培养72h。应用冷无水乙醇于-20℃固定15min，用5％脱脂乳封闭。应用PBST洗涤3次，加入纯化的rHQ06Sw、WH303以及稀释的CSFV阳性和阴性血清(100μL/孔)，于37℃孵育1h。应用PBST洗涤3次，每孔加入100μL HRP标记的山羊抗鼠IgG(catalog no.A3682；Sigma)和兔抗猪IgG(H&L)(catalog no.ab6777；Abcam)(1:2 000)孵育60min。应用PBST洗涤3次，加入50mL的AEC显色。于37℃孵育10min后，弃掉AEC，应用PBS洗涤1次，利用光学显微镜(Nikon公司)观察结果。
1.8Western blotting检测rHQ06Sw与CSFV E2蛋白反应活性
将BHK-21和CHO细胞表达的CSFV石门株E2蛋白、悬浮的HEK293细胞表达的C株E2蛋白以及纯化的CSFV进行SDS-PAGE，将蛋白转印至硝酸纤维素(NC)膜后，用5％脱脂乳于室温孵育2h。应用PBS洗涤1次后，将NC膜分别与HQ06腹水(1:1 000)、WH303(1:1 000)以及纯化的rHQ06Sw孵育1.5h。用PBST洗涤后，分别加入HRP标记的山羊抗鼠(1:1 000)、兔抗猪IgG(1:2 500)孵育1h。再次洗涤完毕后，使用多功能荧光化学发光成像分析系统进行图像扫描。
1.9表面等离子体共振(SPR)试验
应用偶联试剂盒(catalog no.BR100050；GE Healthcare)将CHO细胞表达的E2蛋白(20μg/mL)固定在CM5感应芯片上。利用Biacore T200仪器(GE Healthcare,LittleChalfont,UK)进行SPR测定。将2倍系列稀释(21.875、43.75、87.5、175和350nM)的rHQ06Sw流过芯片表面。应用BIA分析软件分析传感图。
1.10间接ELISA检测rHQ06Sw与BVDV的交叉反应
应用BVDV E2蛋白包被ELISA板，每孔包被100ng。在4℃孵育12h后，应用5％脱脂乳于37℃封闭2h。将纯化的rHQ06Sw、悬浮HEK293的培养上清、BVDV猪源阳性血清和阴性血清进行2倍系列稀释后加入到ELISA反应板中，于37℃孵育1h。应用PBST洗涤3次，加入HRP标记的兔抗猪IgG(H&L)(1:4 000)继续孵育1h，之后加入TMB底物显色，应用2M H2SO4终止反应，检测波长为450nm的吸光度。
1.11病毒中和试验
将PK-15细胞铺于96孔板中，细胞生长至70％时进行试验。病毒中和试验分为两组，第一组为稀释抗体组：将纯化的rHQ06Sw(1mg/mL)2倍系列稀释，做6个稀释梯度和4个重复。将稀释的rHQ06Sw与100TCID50的CSFV等体积混合，于37℃温箱中孵育2h。
第二组为稀释病毒组：将CSFV以10倍系列稀释，稀释为5个梯度4个重复，将稀释的病毒与rHQ06Sw(1mg/mL)等体积混合，在37℃条件下孵育2h。孵育结束后，将rHQ06Sw-CSFV混合物接种96孔板培养的PK-15细胞。孵育3h后，弃去混合液，更换为含有2％胎牛血清的DMEM培养基，继续在37℃温箱中培养72h。
接毒细胞应用冷无水乙醇于-20℃条件下固定15min，然后加入1:100稀释的CSFV阳性血清，于37℃孵育2h后，应用PBS洗涤5次，之后加入1:100稀释的FITC标记的兔抗猪IgG(catalog no.F1638；Sigma-Aldrich)和1:1 000稀释的伊文斯蓝(catalog no.E2129；Sigma-Aldrich)，放入37℃温箱，孵育1h。孵育结束后，用PBS清洗5次，再加入1:1 000稀释的DAPI(catalog no.D9542；Sigma-Aldrich)进行细胞核染色，37℃放置10min，用PBS清洗5次后，应用荧光显微镜(Nikon公司)分析抗体中和效价。
1.12统计学分析
应用GraphPad Prism5.0软件分析所有的数据。误差线表示图中所有平均值(SD)的标准偏差。
2、结果
2.1rHQ06Sw重链、轻链重组质粒的构建
分别以HQ06可变区基因和猪源抗体恒定区基因为模板，利用融合PCR的方法分别扩增rHQ06SwHC和LC基因(重链的核苷酸序列为SEQ ID No.3所示，轻链的核苷酸序列为SEQID No.4所示)，得到预期大小的HC、LC大小分别为1 500bp和750bp(图1A)；重组质粒经BamHI和EcoRI双酶切后在1 500bp和750bp处出现特异条带(图1B)，测序结果显示其与目标序列一致，表明pFUGW-HC和pFUGW-LC质粒构建成功。
2.2rHQ06Sw猪源抗体细胞系的筛选
将HEK293T、CHO和悬浮HEK293细胞表达的猪源rHQ06Sw抗体应用HiTrap Protein AHP预装柱进行纯化，用双抗体夹心ELISA检测抗体与兔抗猪IgG的反应，结果表明，三种细胞表达的抗体均能与兔抗猪IgG反应，其中悬浮HEK293细胞表达的rHQ06Sw与兔抗猪IgG的反应性最好(图2A)，因此选用悬浮HEK293制备rHQ06Sw抗体。本发明进一步利用有限稀释法获得单个细胞克隆，检测这些克隆细胞的活力和表达抗体的能力，结果表明4#细胞克隆表达的抗体与兔抗猪IgG的反应性最好(图2B)。
rHQ06Sw抗体重链的氨基酸序列为SEQ ID No.1所示，轻链的氨基酸序列为SEQ IDNo.2所示。
本发明将稳定表达所述重组猪瘟病毒E2蛋白猪源化单克隆抗体rHQ06Sw的悬浮HEK293细胞的4#细胞克隆提交中国微生物菌种保藏管理委员会普通微生物中心进行保藏，其微生物保藏编号为：CGMCC No.14721。
2.3rHQ06Sw猪源抗体活性的检测
非还原性SDS-PAGE结果表明，rHQ06Sw抗体可成功组装成为160kDa大小的蛋白；还原的SDS-PAGE显示，rHQ06Sw抗体重链为55kDa、轻链为25kDa，与抗体蛋白的分子量一致(图3A和3B)。为了证明rHQ06Sw是否是猪源化抗体，用8％非还原性SDS-PAGE进行Westernblotting分析，在160kDa检测到抗体的目的条带，表明rHQ06Sw可以和抗猪IgG抗体反应(图3C)。rHQ06Sw猪源抗体活性也通过双抗体夹心ELISA得到验证，结果表明，rHQ06Sw与兔抗猪IgG有强烈的反应(图3D)。以上结果证明，rHQ06Sw抗体成功组装，并且可被兔抗猪IgG识别。
2.4rHQ06Sw抗体的反应原性检测
分别用间接ELISA、Western blotting和IPMA验证rHQ06Sw抗体和CSFV E2蛋白的反应性。间接ELISA结果表明，rHQ06Sw抗体与不同表达系统表达的E2蛋白有良好的反应性(图4A)，并且呈剂量依赖性(图4B)。IPMA实验进一步证明了rHQ06Sw可以识别CSFV E2蛋白，具有良好的反应原性(图4C)。Western blotting结果同样表明，rHQ06Sw抗体能够识别不同系统表达的E2(图4D)，并且能与CSFV感染细胞的E2蛋白反应(图4E)，进一步证明rHQ06Sw抗体的特异性。综上所述，rHQ06Sw抗体与CSFV E2蛋白有良好的反应性。
2.5rHQ06Sw抗体与CSFV E2蛋白的结合力分析
应用SPR试验检测了rHQ06Sw抗体与CSFV E2蛋白的结合力。传感图显示，rHQ06Sw抗体可与CSFV E2蛋白相互作用，并且这种作用呈剂量依赖性(图5)，表明rHQ06Sw抗体可以特异性识别并结合CSFV E2蛋白。应用1:1结合模型分析，ka值为9.839×104M-1·s-1，KD值为1.308×10-8M(表2)，表明rHQ06Sw与CSFV E2蛋白具有良好的反应性。以上结果证实，rHQ06Sw抗体与CSFV E2蛋白具有很高的亲和力。
表2 SPR传感图数据
2.6rHQ06Sw抗体与BVDV交叉反应的检测
为了检测rHQ06Sw抗体与瘟病毒属其他成员的交叉反应，本发明应用间接ELISA检测rHQ06Sw抗体与BVDV E2蛋白的反应性。间接ELISA检测结果表明，BVDV E2蛋白与抗BVDV猪源阳性血清有良好的反应性，并且呈剂量依赖性，但不与rHQ06Sw抗体反应。结果表明，rHQ06Sw抗体与BVDV无交叉反应(图6)。
2.7rHQ06Sw对CSFV的中和活性
由于HQ06可以识别CSFV E2蛋白表面的772LFDGTNP778表位(Peng WP,Hou Q,XiaZH,Chen D,Li N,Sun Y,Qiu HJ.2008.Identification of a conserved linear B-cellepitope at the N-terminus of the E2glycoprotein of classical swine fevervirus by a phage-displayed random peptide library.Virus Res135:267–272.)，因此应用中和试验鉴定该抗体中和病毒的能力。将CSFV稀释为100TCID50/50μL，将其与等体积的2倍系列稀释的rHQ06Sw抗体(1mg/mL)进行孵育，然后接种PK-15细胞，于37培养48h，进行间接免疫荧光试验(IFA)。结果表明，在1:2、1:4和1:8rHQ06Sw稀释孔中，未检测到CSFV特异性荧光灶，且在1:16rHQ06Sw稀释孔中，只检测到少量荧光灶，此结果表明，rHQ06Sw效价为1:12，具有中和CSFV的活性(图7A)。
将CSFV石门株(105.5TCID50/mL)以10倍系列稀释，与等体积的rHQ06Sw(1mg/mL)感作后接种PK-15细胞，通过IFA检测重组抗体中和病毒的能力。结果表明，终浓度为0.5mg/mL的rHQ06Sw抗体对300TCID50的CSFV有一定的抑制作用，当CSFV稀释至30TCID50时，rHQ06Sw抗体可以将其完全中和(图7B)。以上结果显示，rHQ06Sw抗体可以有效中和CSFV。
SEQUENCE LISTING
<110> 中国农业科学院哈尔滨兽医研究所
<120> 重组猪瘟病毒E2蛋白猪源化单克隆抗体及其制备方法和应用
<130> HLJ-2001-170517A
<160> 4
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gccaagccat ccgtcttcat cttcccgcca tcgaaggagc agttagagac ccaaactgtc 480
tctgtggtgt gcttgctcaa tagcttcttc cccagagaag tcaatgtcaa gtggaaagtg 540
gatggggtgg tccaaagcag tggcatcctg gatagtgtca cagagcagga cagcaaggac 600
agcacctaca gcctcagcag caccctctcg ctgcccacgt cacagtacct aagtcataat 660
ttatattcct gtgaggtcac ccacaagacc ctggcctccc ctctggtcaa aagcttcagc 720
aggaacgagt gtgaggctta g 741
Claims (10)
1.一种重组猪瘟病毒E2蛋白猪源化单克隆抗体，其特征在于：其重链的氨基酸序列为SEQ ID No.1所示，轻链的氨基酸序列为SEQ ID No.2所示。
2.按照权利要求1所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体，其特征在于：所述重链的编码基因的核苷酸序列为SEQ ID No.3所示，所述轻链的编码基因的核苷酸序列为SEQID No.4所示。
3.一株稳定表达权利要求1所述重组猪瘟病毒E2蛋白猪源化单克隆抗体的悬浮HEK293细胞系，其特征在于，其微生物保藏编号为：CGMCCNo.14721。
4.一种权利要求1或2所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体的制备方法，其特征在于，包括以下步骤：
将重组猪瘟病毒E2蛋白猪源化单克隆抗体的重链和轻链的编码基因分别克隆至真核表达载体，共转染或转化宿主细胞，构建共表达抗体重链和轻链的细胞系；收获所表达的重组抗体，纯化，即得。
5.按照权利要求4所述的制备方法，其特征在于：所述宿主细胞选自HEK293T细胞、CHO细胞或悬浮HEK293细胞中的任意一种，优选为悬浮HEK293细胞；
所述真核表达载体为慢病毒载体；优选的，所述慢病毒载体为pFUGW；
所述重链的编码基因的核苷酸序列为SEQ ID No.3所示，所述轻链的编码基因的核苷酸序列为SEQ ID No.4所示。
6.一种权利要求1或2所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体的制备方法，其特征在于，包括以下步骤：
(1)将重组猪瘟病毒E2蛋白猪源化单克隆抗体的重链和轻链的编码基因分别与慢病毒载体pFUGW可操作的连接，得到含有重链编码基因的重组质粒pFUGW-HC和含有轻链编码基因的重组质粒pFUGW-LC；(2)将重组质粒pFUGW-HC以及辅助质粒共转染细胞，得到包装重组质粒pFUGW-HC的慢病毒；将重组质粒pFUGW-LC以及辅助质粒共转染细胞，得到包装重组质粒pFUGW-LC的慢病毒；(3)将包装重组质粒pFUGW-HC的慢病毒和包装重组质粒pFUGW-LC的慢病毒共转导细胞，得到共表达抗体重链和轻链的细胞系；收获所表达的重组抗体，纯化，即得。
7.按照权利要求6所述的制备方法，其特征在于：步骤(1)所述重链的编码基因的核苷酸序列为SEQ ID No.3所示，所述轻链的编码基因的核苷酸序列为SEQ ID No.4所示；
步骤(2)所述辅助质粒包括pMD2.G和psPAX2；步骤(2)所述细胞为HEK293T细胞；
步骤(3)所述细胞选自HEK293T细胞、CHO细胞或悬浮HEK293细胞中的任意一种，优选为悬浮HEK293细胞。
8.权利要求1或2所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体在制备猪瘟病毒诊断制剂、治疗制剂或者猪瘟病毒抗体检测试剂中的应用。
9.权利要求3所述的悬浮HEK293细胞系在制备猪瘟病毒诊断制剂、治疗制剂或者猪瘟病毒抗体检测试剂中的应用。
10.一种用于猪瘟病毒抗体检测的竞争ELISA试剂盒，其特征在于，包括：HRP标记的权利要求1或2所述的重组猪瘟病毒E2蛋白猪源化单克隆抗体。
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