猪巨细胞病毒gB蛋白抗原表位富集区的表达和抗原性的分析  

Expression and antigenic analysis of the immunodominant region of porcine cytomegalovirus gB protein

作　　者：李晶[1] 余兴龙[1] 李润成[1] 黄泽彬[1] 葛猛[1] 向卫军[1] 李薇[1] 王亚[1] 

机构地区：[1]湖南农业大学动物医学院,湖南长沙410128

出　　处：《中国预防兽医学报》2010年第2期126-130,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：湖南省科技重大专项(2007FJ1003);湖南省农业大学人才引进基金(04YJ103)

摘　　要：为表达猪巨细胞病毒(PCMV)gB基因,本研究根据GenBank登录的PCMV gB基因序列设计1对引物,以感染PCMV猪肺细胞总DNA为模板,采用PCR扩增得到gB基因片段,克隆于pMD-18T并进行核苷酸序列测定。利用DNAStar分析gB蛋白的抗原表位,选择抗原表位富集的2个基因片段(命名为PCMVgB1和PCMVgB2)分别克隆到原核表达载体pET-32a(+)中,构建重组表达质粒并转化Rosetta(DE3)宿主菌。结果显示:扩增的gB基因与GenBank登录的PCMV gB基因的核苷酸同源性在97.6%～98.9%之间,推导的氨基酸同源性在97%～98.6%之间。经IPTG诱导的含pET-gB1和pET-gB2重组质粒的宿主菌可表达重组gB1和gB2蛋白,SDS-PAGE显示分子量约为62ku和36ku。Western blot和间接ELISA结果表明,重组gB1和gB2蛋白均具有反应原性。The glycoprotein B （gB） gene encoding structural protein of porcine cytomegalovirus （PCMV） was amplified by PCR using primers derived from the published PCMV gB gene sequence, cloned and sequenced. The gB gene shared 97.6 %-98.9 % sequence identity at nucleotide level and 97 %-98.6 % identity at amino acid level with PCMV gB reference genes in GenBank. Two immunodominant regions of PCMV gB1 and PCMV gB2 were identified by DNAStar and were subcloned into prokaryotic expression vector pET-32a（＋）. The resulting plasmids of pET-gB1 and pET-gB2 were transformed into E.coli competent Rosetta, respectively, and induced by IPTG. Recombinant protein of 62 kua （gB1） protein and a 36 kua （gB2） protein were expressed and detected by SDS-PAGE. Western blot and indirect ELISA analysis showed that both recombinant proteins could specifically react with antiserum against PCMV.

关 键 词：猪巨细胞病毒 GB基因 原核表达 间接ELISA 

分 类 号：Q786[生物学—分子生物学]