Construction and expression of eukaryotic expression vector pIHSP65 of mycobacterium tuberculosis

Objective To construct the eukaryotic expression vector based on HSP65 gene of mycobacterium tuberculosis.Methods HSP65 gene was cloned from genome of mycobacterium tuberculosis H37Rv strain by PCR,and inserted into multiple cloning site A's corresponding sites of dicistronic eukaryotic expression vector pIRES to construct eukaryotic expression vector pIHSP65,then transfected into Hela cell by using polycation.Finally,the expression of HSP65 protein was detected by immunochemical staining.Results The length and sequence of the cloned HSP65 segment were correct.The positive expression of HSP65 in Hela cell,which had been transfection with recombinant plasmid,was identified by immunohistochemical method.Conclusion The successful construction of eukaryotic expression vector pIHSP65 and effective expression in Hela cells,lays the foundation for constructing two-antigens dicistronic eukaryotic expression plasmid and further study in animal.