Expression And Purification Of Mycobacterium Tuberculosis Ag85B/human IL-2 Fusion Protein And Research Of Its Immunotherapy Effects On Bladder Tumor

Bladder tumor is the most common tumor in urinary system,which has the clinical characteristic of high incidence rate, high recurrence rate and being difficult to treat . The low immunological function of bladder mucous membrane is one reason of the remaining tumor cells escaping immune surveillance and recurring ,then how to raise the immunological function of bladder patients is the key to treat and prevent recurrence of bladder tumor. It was reported that intravesical instillation of Bacillus Caimette-Guerin (BCG) is one of the most effective way of treatment and preventing recurrence of baldder cancer ,and the clinical results would be significantly improved when intravesical perfusion of BCG combined interleukin 2 (IL-2). However, both BCG and IL-2 being applied to treat bladder cancer has the shortage of large dose ,long course of therapy and more side effects, so it is important to explore a more effective and safe immunotherapy approach of bladder cancer.Ag85B, which is the main excreting protein and cross reactive protein in BCG, Mycobacterium tuberculosis and other Mycobacteriums , and as the main biological component of BCG playing effect in vitro and in vivo, has the ability of inducing fierce Th1 immunologic reaction. Utilization of its immunogenicity , Ag85B plays an important role not only in making polypeptide vaccine and gene vaccine in tuberculosis prophylaxis ,but also in treatment of bladder cancer and other tumor in recent years. In the present study , mycobacterium tuberculosis Ag85B gene and human IL-2 cDNA were spliced to Ag85B/IL-2 fusion gene by genetic recombination technology. After the Ag85B/IL-2 fusion protein was prokaryotic expressed and purified ,its immunotherapy effects on bladder tumor in vitro and in vivo had been studied .This has established a groundwork for the further exploiture and application of Ag85B/IL-2 fusion protein. The main results are as follows: 1. Construction of mycobacterium tuberculosis Ag85B/IL-2 fusion protein expression vector and prokaryotic expression ⑴ Firstly, by way of PCR, Mycobacterium tuberculosis Ag85B gene was amplified with plasmid pUC18/Ag85B template , and cloned into plasmid pUC18 at the restriction endonuclease sites of EcoRI and KpnI. The recombinant plasmid was named as pUC-Ag85B . Secondly, human IL-2 cDNA was acquired by PCR amplification with template of pCIIL-2 and inserted into plasmid pUC-Ag85B at the restriction endonuclease sites of KpnI and BamHI, which located at downstream of Ag85B gene through the linker sequence coding Gly4Ser1. The recombinant which was named as pUC-Ag85B/IL-2 was identified by restriction enzyme analysis and DNA sequencing analysis. The results showed that Ag85B/IL-2 fusion gene was constructed successfully.⑵ To construct expression vector pGEX-Ag85B/IL-2, pTHio-Ag85B/IL-2 and pBV-Ag85B/IL-2, Ag85B/IL-2 was subcloned into three different plasmids pGEX4T-1,pThioHis,pBV220 from pUC-Ag85B/IL-2, and the results were identified by restriction enzyme analysis. After three recombinants being transform into host E.coli BL21,DH5αand Top10, respectively, engineering E.coli BL21/ pGEX-Ag85B/IL-2,Top10/ pTHio-Ag85B/IL-2 and DH5α/ pBV-Ag85B/IL-2 were successfully constructed . ⑶To express the recombinant Ag85B/IL-2 fusion protein, BL21/ pGEX-Ag85B/IL-2 and Top10/ pTHio-Ag85B/IL-2 were induced by IPTG, and DH5α/ pBV-Ag85B/IL-2 was induced at 42℃. The results revealed that only BL21/ pGEX-Ag85B/IL-2 expressed a new protein about molecular weight of 66kD , which was similar to the theory molecular weight of GST-Ag85B/IL-2, and its amount reached to about 31% of the total bacteria protein. By western blot with rabbit anti-human IL-2 momoclonal antibody, the new 66kD protein was positive. This indirectly confirmed that it was the anticipated fusion protein GST-Ag85B/IL-2. ⑷ Under the induction of IPTG at 37℃for 5-6 h, the expression amount of GST-Ag85B/IL-2 reached to the peak. There was no obvious difference in expression amount with IPTG concentration from 0.1 mmol/L to 1.5 mmol/L. So the optimize...