Cloning, Expression and Purification of Mycolyltransferase C of Mycobacterium tuberculosis in prokaryotic host

Objective: Antigen ۸۵ complex of Mycobacterium tuberculosis includes three immunogenic proteins which are important TB vaccine candidates. The use of these protein as a component of subunit vaccines, as a part of DNA vaccines or recombinant BCG boosters, enhances their recombinant production. Recombinant production of these mycobacterial proteins located in the cell wall somehow differs from the other proteins, as they are partially apolar. Therefore, this study aims to produce recombinant Ag۸۵C as a vaccine candidate. Methods: Ag۸۵C gene was cloned in pJET۱.۲ and subsequently in pET۳۲a(+). Both recombinant plasmids were sequenced. Expression of the recombinant protein was induced with ۱mM IPTG. Recombinant Ag۸۵C was purified through dissolving the inclusions in ۸M urea buffer, absorbed to Ni-NTA resins, washed by buffers with decreasing urea concentrations, and finally eluted in aqueous solution. Western blot analysis was performed using anti-۶His tag antibody, rabbit anti-M. tuberculosis polyclonal antibody, and the serum from hospitalized TB patients. Results: Ag۸۵C successfully cloned in both plasmid vectors. The recombinant Ag۸۵C expressed in the E. coli host and was purified with significant yield. Conclusion: Western blot results along with that of sequencing ensure accurate production of recombinant Ag۸۵C, retaining its partial epitopes

کلیدواژه ها:

Ag۸۵ complex ، Ag۸۵C ، TB vaccine ، Mycobacterium tuberculosis ، مایکوباکتریوم توبرکلوزیس ، کمپلکس آنتیژن ۸۵ ، آنتیژن ۸۵C واکسن سل