The Preparation of Mycobacterium tuberculosis HSP65 DNA Vaccine and the Prokaryotic Expression of the Recombinant HSP65 Protein

The purpose of this study is to construct DNA Vaccine of the Mycobacterium tuberculosis 65 ku Heat shock protein and clone it into prokaryotic expression vector to express, because it is a very important antigen against tuberculosis infection. The method: The HSP65 gene is amplified from Mycobacterium tuberculosis H37Rv genome DNA using PCR method. The PCR product is digested by the restriction endonucleases and then ligated to the eukaryotic expression vector pJW4303 which is digested by the same restriction endonucleases. The recombinant plasmid is called pJW-HSP65. Meantime, the HSP65 gene is inserted into the prokaryotic expression vector pET-22b(+) to product the recombinant prokaryotic expression vector pET22b-HSP65. Plasmids containing the right insertion were sequenced to confirm their identity. Transform the BL21(DE3)/PolysS strain with this recombinant prokaryotic expression vector,and induce it with IPTG. The HSP65 protein is highly expressed. After purification, the expressed protein can be used as protective Mycobacterium Tuberculosis antigen to detect the immunity efficiency of the HSP65 DNA Vaccine.In conclusion, we have successfully constructed HSP65 DNA vaccine and its protein has been expressed.