牛呼吸道合胞体病毒G蛋白的截短表达与鉴定  

Expression and Identification Truncated Glycoprotein G of Bovine Respiratory Syncytial Virus in Escherichia coli

作　　者：冯军科[1] 薛飞[1] 李娇[1] 祖立闯[1] 朱远茂[1] 任宪刚[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/大动物传染病研究室,哈尔滨150001

出　　处：《中国生物工程杂志》2008年第12期24-29,共6页China Biotechnology

基　　金：黑龙江省科技攻关项目(GA06B202-3)

摘　　要：将牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)G基因截短成2个片段G1和G2,然后以人工合成的牛呼吸道合胞体病毒G基因为模板,利用PCR分别扩增G1和G2基因片段,其大小分别为570 bp和308 bp。将目的片段定向克隆到pET30a表达载体,酶切及测序鉴定均正确后,转化BL21表达菌,经IPTG诱导后G1和G2基因片段均获得了表达,且为可溶性表达。利用Ni柱亲和层析法在非变性条件下纯化重组蛋白,经免疫印迹试验鉴定证明纯化的重组蛋白G1具有良好的抗原性和特异性,而重组蛋白G2无反应性。应用纯化的重组蛋白G1进行的间接ELISA与免疫印迹试验在国内牛血清中检测到了BRSV血清抗体。对建立BRSVG蛋白的血清学诊断方法和为开展BRSVG蛋白生物学功能的研究奠定了基础。Two fragments G1 and G2 of the glycoprotein G gene of bovine respiratory syncytial virus （BRSV） were selected for expression in Escherichia coli based on the analysis of glycoprotein G by DNA Star software. Then the two fragments of glycoprotein G were amplified by PCR with synthesized G gene of BRSV as the template. The amplified fragments G1 and G2 are 570bp and 308bp in length, respectively. The PCR products were cloned into pET30a vector and expressed in soluble form in E. coli after induction of cultured E. coli with IPTG. Both of the recombinant proteins G1 and （;2 were purified by immobilized Ni ion affinity chromatography under native conditions. Then the purified proteins were analysed by Western blotting. The results showed that the purified recombinant protein G1 retained good antigenicity and specificity. But the purified recombinant protein G2 didn＇ t possess biological activity. Antibodies against BRSV were detected in suspected bovine serum samples in China by using indirect ELISA and Western blotting with the purified recombinant protein G1. The purified recombinant protein G1 might be used as antigen for establishing serological methods for diagnosis of BRSV infection. And the purified recombinant protein G1 might also be used for preparing polyclonal and monoclonal antibodies for research on biological functions of glycoprotein G of BRSV.

关 键 词：牛呼吸道合胞体病毒 G基因 克隆 表达 

分 类 号：Q819[生物学—生物工程]