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  

Expression of the truncated E2 protein-encoding gene of classical swine fever virus in Escherichia coli and preparation of a monoclonal antibody against E2 protein

作　　者：侯强[1] 彭伍平[1] 孙元[1] 仇华吉[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：《中国兽医科学》2008年第1期1-5,共5页Chinese Veterinary Science

基　　金：国家重点基础研究发展规划(973)项目(2005CB523202)

摘　　要：利用RT-PCR扩增了猪瘟病毒(CSFV)兔化弱毒疫苗株(C株)的E2蛋白主要抗原区(tE2)编码区,并定向克隆到表达载体pPROEX-HTb中,获得重组表达载体pPROEX-tE2,将其转化大肠杆菌DH5α菌株,经IPTG诱导,tE2蛋白获得高效表达;经检测,该重组蛋白能被CSFV C株抗血清识别。用纯化的重组蛋白免疫BALB/c小鼠,取其脾淋巴细胞与SP2/0骨髓瘤细胞融合,获得了1株稳定分泌抗tE2蛋白单克隆抗体的杂交瘤细胞株。经检测,该单克隆抗体能够与CSFV C株和石门株以及杆状病毒表达的重组E2蛋白发生特异性反应。推测,该单克隆抗体是针对CSFV E2蛋白的一个保守线性表位。A truncated gene encoding the major antigenic domains of E2 protein of classical swine fever virus（CSFV） was amplified by RT PCR from the genomic RNA of CSFV C-strain and cloned into pPRO EX-HTb expression vector to obtain recombinant pPROEX-tE2. The truncated E2 protein（tE2） was expressed with high level in pPROEX tE2 transformed Escherichia coli cells after induction with IPTG. The recombinant protein could be recognized by CSFV C-strain antisera in either Western-blotting or ELISA. The tE2 protein purified using a Ni-chelating HisTrap affinity column was used to immunize BALB/c mice,of which splenocytes were fused with SP2/0 cells with PEG3250 after the 3rd immunization. A hybridoma cell line stably secreting a monoclonal antibody（McAb） directed against the tE2 protein was screened by ELISA. The McAb was able to react specifically with C-strain and Shimen strain,and recognized a linear conserved epitope on the E2 protein. The McAb was identified to be IgG1 subtype.

关 键 词：猪瘟病毒 E2蛋白 原核表达 单克隆抗体 

分 类 号：S852.659.6[农业科学—基础兽医学]