Cloning Of ESAT-6 Gene Of Mycobaterium Tuberculosis And Its Expresson In Mycobacterium Smegmatis Mc~2 155 And Pichia Pastoris

Objective [1] to construct the E.coli-Mycobacterium shuttle plasmid containing esat-6 gene of Mycobaterium tuberculosis, express in Mycobacterium smegmatis mc2 155 and the plasmid was transformed into BCG by electroporation. [2] To construct the recombinant plasmid containing ESAT-6 gene,express in the yeast Pichia pastoris, with a view to provide the experimental reference and guideline for the development of new vaccines of tuberculosis.Methods [1] based on the reported sequences of the ESAT-6 and the shuttle vector ps3000 one pair of primers was designed. Using PCR technique, the esat-6 gene was amplified and inserted into cloning vector pGEMT, the recombinant was identified by PCR, restriction endonucleases digestion and DNA sequenceing.[2]after subcloning the shuttle plasmid ps3000- ESAT-6, the shuttle plasmids was transformed into BCG and Mycobacterium smegmatis mc2 155 by electroporation. The activity of the target prtein was by SDS-PAGE and immunoblotting. [3] based on the reported sequences of the esat-6 and the shuttle vector pPICZaA one pair of primers was designed. Using PCR technique, the ESAT-6 gene was amplified and inserted into cloning vector pGEMT, the recombinant was identified by PCR, restriction endonucleases digestion and DNA sequenceing. [4] After subcloning the shuttle plasmid pPICZaA-ESAT6, the shuttle plasmids was transformed and integrated into GS115. To analyze Pichia integrants and selected multiple gene insertion Zeocin transformants by PCR. The expressed protein was identified by SDS-PAGE and western-blot.Results [1] The ESAT-6 gene was amplified from the genomic DNA by polymerase chain reaction. Shuttle vector ps3000-ESAT-6 was correctly construced. The target protein could be expressed and recognized by the mice antiserum to the purified protein pET-ESAT-6, which demonstrated that the recombinant M.smegmatis mc2 155 could expressed the target protein that has biologic activity. [2] Shuttle vector pPICZaA-ESAT-6 was correctly construced. SDS-PAGE can reveal the intracellular expression, a-f?actor signal sequence and C-terminal tag will add 12KDa to the size of my protein which can be recognized by the serum of active tuberculosis patients.Conclusion [1]The recombinant M.smegmatis mc2 155 was successfully constructed and pave the way for further study of recombinant BCG vaccine that expressed ESAT-6 protein.[2]Using Pichia pastoris expression system express the intracellular esat-6 protein and provide the basis to develop a new tuberculous vaccine.