Construction of Mycobacterium bovis ag85b Gene Protokaryotic Expression Vector

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 recombinant 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.