鸡传染性喉气管炎病毒gB基因的克隆及其在耻垢分枝杆菌中的表达

以ILTV基因组为模板 ,利用PCR特异扩增出gB基因 ,定向克隆到中间质粒载体pY_α ,构建了中间质粒pY_α_gB。然后以中间质粒pY_α_gB为模板 ,扩增出含有人结核分枝杆菌启动子hsp70基因和堪萨斯分枝杆菌α信号肽基因的hsp_α_gB片段 ,回收补平后与穿梭表达载体pRR3平端连接 ,从而构建大肠杆菌_分枝杆菌穿梭表达质粒pR_α_gB。再将其电转化至耻垢分枝杆菌M .smegmatismc2 15 5 ,ELISA检测表明重组菌株M .smegmatismc2 15 5 (pR_α_gB)的表达产物具有很好的反应原性。Westernblot检测说明gB基因在分枝杆菌中获得了表达并具有良好的免疫原性。鸡胚中和试验结果表明该重组菌株可以中和 1个剂量EID50 的ILTV强毒 ,能够保护SPF鸡胚抵抗强毒攻击

Cloning and Expression of gB Gene of Infectious Laryngotracheitis Virus in M.Smegmatis

Firstly, the complete gB gene of a domestic isolation stain were amplified by PCR, and the 2.6 kb gene fragment was obtained. Then the recombinant plasmid pY-alpha-gB was constructed by cloning PCR product into pY-alpha vector, and the shuttle expression plasmid pR-alpha-gB was constructed by cloning the hsp-alpha-gB gene into the downstream sequences of pRR3 vector. The recombinant plasmid was identified by restriction enzyme digestion and the sequence analysis, which was electrophoreted into M. smegmatis mc2 155. At last, the expressed gB proteins were successfully detected by ELISA and Western blot, which seems to be immunogenic crucially. The recombinant bacterial stain M. smegmatis mc2 155 (pR-alpha-gB)could protect SPF chick embryo from one lethal dose of ILTV.