A群猪轮状病毒VP7蛋白单克隆抗体的制备及鉴定

中国畜牧兽医

A群猪轮状病毒VP7蛋白单克隆抗体的制备及鉴定

迟延彬^1，2，陈建飞^{2，时洪艳²，张鑫²，石达²，李长龙²，韩斅²，陈洪岩^1，2，冯力^{1^，2}}

（1.东北农业大学生命科学学院，黑龙江哈尔滨 150030；2.中国农业科学院哈尔滨兽医研究所，兽医生物技术国家重点实验室，猪传染病研究室，黑龙江哈尔滨 150001)

收稿日期:2013-06-26 出版日期:2014-02-20 发布日期:2014-03-27
通讯作者: 冯力，男，辽宁人，博士生导师，研究员，研究方向：兽医微生物学及分子生物学。E-mail：fengli_h@163.com
作者简介:迟延彬(1989—)，男，黑龙江人，硕士生，研究方向：兽医微生物学及分子生物学。
基金资助:
科技部农业科技成果转化资金（2011GB23260003）猪病毒性腹泻三联活疫苗的中试及应用；黑龙江省高等学校科技创新团队项目（2011TD001）猪病毒性腹泻疾病防治。

Preparation and Identitficaion of Monoclonal Antibody against VP7 Protein of Group A Porcine Rotavirus

CHI Yan-bin¹^，2，CHEN Jian-fei²，SHI Hong-yan²，ZHANG Xin²，SHI Da²，LI Chang-long²，HAN Xiao²，CHEN Hong-yan¹^，2,FENG Li¹^，2

（1.College of Life Science，Northeast Agricultural University，Harbin 150030，China;2.Division of Swine Infectious Diseases，State Key Laboratory of Veterinary Biotechnology，Harbin Veterinary Research Institute，Chinese Academy of Agricultural Sciences，Harbin 150001，China）

Received:2013-06-26 Online:2014-02-20 Published:2014-03-27

摘要/Abstract

摘要： 试验旨在制备A群G11血清型猪轮状病毒（porcine rotavirus，PoRV）VP7蛋白的单克隆抗体，并鉴定其免疫学特性。将A群G11血清型PoRV VP7基因克隆至pGEX-6P-1载体，然后转化大肠杆菌DH5α感受态细胞，IPTG诱导表达GST-VP7融合蛋白。分离纯化融合蛋白GST-VP7并免疫BALB/c小鼠，取其脾细胞与骨髓瘤细胞SP2/0融合，利用间接ELISA法筛选阳性杂交瘤细胞并进行有限稀释法克隆化筛选。经3次筛选，最终得到1株能与GST-VP7蛋白反应的特异性、稳定分泌抗体的阳性细胞克隆。Western blotting鉴定结果显示该单克隆抗体具有良好的免疫原性及特异性；MTT法中和试验结果显示其具有中和活性；分泌的单克隆抗体亚型为IgG2b。本试验结果表明A群PoRV VP7蛋白在大肠杆菌中成功表达且制备了单克隆抗体，可用于PoRV VP7蛋白结构和功能的研究，也为猪轮状病毒病的预防、诊断和治疗等研究奠定基础。

关键词: A群猪轮状病毒; VP7基因; 单克隆抗体

Abstract: To prepare the monoclonal antibodies against VP7 protein of group A porcine rotavirus (PoRV) serotype G11,VP7 gene of PoRV serotype G11 was cloned into the vector pGEX-6P-1. The recombinant plasmid was transformed into E.coli competent cells DH5α and induced by IPTG. Hybridomas were produced by fusing SP2/0 cells with spleen cells from mouse immunized with GST-VP7 recombinant protein. One hybridomas secreting MAb against VP7 protein was identified by indirect ELISA. Western blotting analysis showed that the MAb could recognize the recombinant VP7 protein and authentic VP7 protein of PoRV，and the specific immunoflurescence was detected in PoRV infected MA104 cells by indirect immunoflurescence assay.The result of MTT method showed that the MAb had the neutralizing activity. The subtype of the MAb was IgG2b. The results showed that VP7 protein was successfully expressed in E.coli， one MAb was preparated and identified，and it could be used for further study of prevention，diagnosis and treatment of porcine rotavirus disease.

Key words: group A procine rotavirus; VP7 gene; monoclonal antibodies

迟延彬，陈建飞，时洪艳，张鑫，石达，李长龙，韩斅，陈洪岩，冯力. A群猪轮状病毒VP7蛋白单克隆抗体的制备及鉴定[J]. 中国畜牧兽医.

CHI Yan-bin，CHEN Jian-fei，SHI Hong-yan，ZHANG Xin，SHI Da，LI Chang-long，HAN Xiao，CHEN Hong-yan,FENG Li. Preparation and Identitficaion of Monoclonal Antibody against VP7 Protein of Group A Porcine Rotavirus[J]. .

参考文献

1 丛文娟，杨继红，邓妍，等.轮状病毒WH-2株VP7基因的原核表达及单克隆抗体的制备[J].细胞与分子免疫学杂志，2011，27(3)：294～296.
2 刘钊，杨占秋，肖红，等.MTT 法在抗病毒药物筛选中的应用[J].武汉大学学报(医学版)，2004，25(3)：332～334.
3 刘勇.轮状病毒 VP7 基因修饰表达及免疫原性研究[D].北京：中国协和医科大学，2000.
4 张志榜，陈建飞，时洪艳，等. 猪流行性腹泻病毒M蛋白单克隆抗体的制备及鉴定[J].中国预防兽医学报，2011，33(7)：568～570.
5 胡小华，李刚，史利军，等. 猪圆环病毒2型间接ELISA抗体检测试剂盒的研制及初步应用[J].中国畜牧兽医，2011，38(3)：199～203.
6 高慎阳，査恩辉，王坤，等.一种“高性价比”切胶纯化原核表达蛋白的方法[J].中国农学通报，2010，26(22)：24～26.
7 童光志.动物传染病学[M].北京：中国农业出版社，2008.
8 Aoki S T,Settembre E C,Trask S D,et al.Structure of rotavirus outer-layer protein VP7 bound with a neutralizing Fab[J]. Science,2009,324(5933):1444～1447.
9 Aoki S T,Trask S D,Coulson B S,et al. Cross-linking of rotavirus outer capsid protein VP7 by antibodies or disulfides inhibits viral entry[J]. Journal of Virology,2011,85(20): 10509～10517.
10 Clarke M L,Lockett L J,Both G W,et al.Membrane binding and endoplasmic reticulum retention sequences of rotavirus VP7 are distinct: Role of carboxy-terminal and other residues in membrane binding[J].Journal of Virology, 1995,69(10): 6473～6478.
11 Doronin K K,Iurov G K,Grinenko N F,et al. Expression of a gene encoding the porcine rotavirus VP7 capsid using a nondefective recombinant adenoviral genom[J]. Mol Gen Mikrobiol Virusol,1995(4):35～39.
12 Ludert J E,Ruiz M C,Hidalgo C,et al. Antibodies to rotavirus outer capsid glycoprotein VP7 neutralize infectivity by inhibiting virion decapsidation[J]. Journal of Virology,2002,76(13): 6643～6651.
13 Manuja B K,Prasad M,Gulati B R,et al. Comparative efficacy of immunological， molecular and culture assays for detection of group A rotavirus from faecal samples of buffalo (Bubalus bubalis) calves[J]. Tropical Animal Health and Production,2010,42(8): 1817～1820.
14 Wang L,Huang J A,Phelps A,et al. Periplasmic expression of part of the major rotavirus capsid protein VP7 containing all the three antigenic regions in Escherichia coli[J]. Gene,1996,177(1～2): 155～162.