猪胸膜肺炎放线杆菌APXⅡA基因原核表达载体的构建及其表达

Expression vector construction and protocaryon expression of Actinobacillus pleuropneumoniae APXⅡA gene

经PCR从含有APXⅡA基因片段的重组质粒 pT APXⅡA中扩增出了APXⅡA基因。同时对目的基因和表达质粒 pGEX 4T 1进行了双酶切 ,连接、转化受体菌 ,经PCR、酶切和序列分析 ,证明连接向位和阅读框架正确。成功构建了APXⅡA基因的原核表达载体 pGEX APXⅡA。重组质粒转化表达菌在IPTG诱导下 ,用SDS PAGE分析表达目的蛋白。结果表明 ,蛋白质的分子质量为 10 2 .5ku。

The APXⅡA gene was amplified from recombinant plasmid pT-APXⅡA by PCR. The gene and expression vector pGEX-4T-1 were digested by restriction endonuclease. The two genes were ligated and transformed into E.coli. It was confirmed by PCR ,restriction endonuclease digestion and sequencing that the orientation and coding frame were correct. The result showed that the recombinant pGEX-APXⅡA had been constructed. The transformed recombinant plasmids were induced by IPTG and the expressed proteins were valued by SDS-PAGE with expected molecular size of 102.5 ku.