猪繁殖与呼吸综合征病毒ORF3基因的表达及其单克隆抗体的制备  

Prokaryotic expression and development of monoclonal antibody against glycoprotein 3(GP3) of PRRSV CH-1a strain

作　　者：刘金霞[1] 周艳君[1] 安同庆[1] 仇华吉[1] 王云峰[1] 童光志[1] 

机构地区：[1]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,黑龙江哈尔滨150001

出　　处：《中国兽医学报》2007年第5期609-612,共4页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(30470072);国家重点基础研究发展规划"(973)"资助项目(2005CB523200)

摘　　要：将猪繁殖与呼吸综合征病毒(PRRSV)CH-1a株的GP3蛋白基因进行截短修饰后,克隆于pGEX6p-1载体中,转化大肠杆菌,并进行诱导表达。经SDS-PAGE检测,融合表达的蛋白rtGP3大小约40 000;Western blotting分析证实,表达的融合蛋白rtGP3能被PRRSV阳性血清所特异性识别。收获融合表达的rtGP3,免疫BALB/c小鼠,取脾细胞与SP2/0骨髓瘤细胞进行融合,分别以表达的融合蛋白rtGP3和GST蛋白作为包被抗原,通过间接ELISA方法对融合细胞的上清液进行检测,结果获得了5株能稳定分泌抗rtGP3蛋白抗体的杂交瘤细胞株,命名为31A2,31G11,32B12,32D4和32H7。利用PRRSV感染的Marc-145细胞进行间接免疫荧光检测显示,所获得的5株单克隆抗体均能与PRRSV感染细胞产生特异性免疫荧光。亚型鉴定结果表明,5株单克隆抗体均为IgG1型,且轻链均为κ链。The ORF3 gene of PRRSV strain CH-1a was truncated and cloned into a prokaryotic expression vector pGEX-6p-1, and a fusion-expressed protein rtGP3 of 40 000 was obtained in E. coll. The protein rtGP3 showed a strong reaction to the PRRSV- positive sera in Western blotting. BALB/c mice were immunized intraperitoneally with purified rtGP3 protein, Murine myeloma cells were fused with the splenocytes of the immunized mice after the third immunization. An indirect ELISA coated with rtGP3 and GST was used to screen hybridomas for production of specific antibody in hybridoma culture fluid. Five hybridomas clone which produced McAbs steadily was obtained, named 31A2, 31G11, 32B12, 32D4 and 32H7. The McAbs showed strong reactivity in IFA on the PRRSV infected Marc-145 cells.The McAbs belong to IgG1 isotype, and their light chain are κ chain,which was isotyped by ELISA using a commercial kit. The McAbs against PRRSV GP3 protein could be used for further analysis of the structure and function of PRRSV.

关 键 词：猪繁殖与呼吸综合征病毒 rtGP3 单克隆抗体 

分 类 号：S585.265[农业科学—作物学]