牛分支杆菌αg85b基因原核表达载体的构建

摘要

To construct a expression recombinant plasmid pET-32a-85b of Mycobacterium bovis ag85b gene,ag85b gene of Mycobacterium bovis was amplified (PCR method) from the Mycobacterium bovis AF2122/97, the ag85b contained 978 bp.Amplification product and vector pET-32a were cut by endonuclease EcoR Ⅰ and Sal Ⅰ. Then the two restriction products were linked together by T4 DNA ligase for cloning ag85b gene into vector pET-32a to construct recombinant plasmid. The recombinant plasmid was transformed into E. coli DH5α. Firstly,extractive recombinant plasmid was cut by double cloning enzymes EcoR Ⅰ and Sal Ⅰ . Secondly,recombinant plasmid was checked by PCR amplification. Lastly,sequencing of recombi nant plasmid was done. The cutting product size and amplification product size were in accord with anticipation,sequencing of recombinant plasmid showed that sequence of amplification gene ag85b was same with the sequence from GenBank. The ag85b gene was successfully amplified and cloned into pET-32a expression vector.%为构建牛分支杆菌αg85b基因的重组表达质粒pET-32a-ag85b,采用聚合酶链反应(PCR)从牛分支杆菌AF2122/97基因组DNA中扩增出αg85b基因(978 bp),然后对扩增产物和载体pET-32a以核酸内切酶EcoR Ⅰ及Sal Ⅰ分别进行双酶切;将两种酶切产物以T4 DNA Ligase连接,将靶基因克隆入载体pET-32a,构建重组质粒.将此重组质粒转化人大肠杆菌DH5α,抽提重组质粒首先经EcoR Ⅰ及Sal Ⅰ双酶切检验,再进行PCR扩增鉴定,最后测序鉴定.酶切片段及PCR扩增片段大小均与预期相符,测序结果与GenBank登录序列完全相同.结果表明,成功地克隆并构建了αg85b基因重组表达质粒pET-32a-ag85b.