A群猪轮状病毒VP7蛋白单克隆抗体的制备及鉴定  

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

作　　者：迟延彬 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] 

机构地区：[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

出　　处：《中国畜牧兽医》2014年第2期70-74,共5页China Animal Husbandry & Veterinary Medicine

基　　金：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

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PoRVVP7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。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 DH5a 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 pro tein of PoRV, and the specific immunoflurescence was detected in PoRV infected MA104 cells by indirect imnmnoflurescence as- say. 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 {or further study of prevention,diagnosis and treatment of porcine rotavirus disease.

关 键 词：A群猪轮状病毒 VP7基因 单克隆抗体 

分 类 号：Q813.2[生物学—生物工程]