Construction Of Prokaryotic Expression Vector Of MTB Lhp-esat6 Fusion Gene And Preliminary Application Of Recombinant CFP10-ESAT6 Fusion Protein

The study aimed to construct the prokaryotic expression vectors carrying MTB esat6 and lhp and lhp-esat6 fusion genes amplified by PCR, and express them in E.coli respectively. The recombinant protein was identified and purified from the expression E.coli. The potential value of the rCFP10-ESAT6 fusion protein in immunodiagnosis for tuberculosis was evaluated by zoopery and clinical assay. PART Ⅰ CONSTRUCTION AND EXPRESSION OF THE PROKARYOTIC EXPRESSION VECTOR OF MTB esat6 GENEObjective: To construct the prokaryotic expression vector carrying MTB esat6 gene and express it in E.coli, and to purify the rESAT6 with affinity chromatography technique.Methods: The esat6 gene fragment with HindIII and BamHI sites was amplified by PCR from MTB genome, then cloned into pQE30 plasmid, sequenced and cloned into pET32a(+) plasmid. The two kinds of prokaryotic expression vectors were transformed into DH5αandBL21(DE3) E.coli respectively, and the esat6 gene was expressed in the presence of IPTG. The rESAT6 was identified by SDS-PAGE and purified with Ni-NTA column. The antigenicity of rESAT6 was confirmed by Western blot.Results: The pQE30-ESAT6 and the pET32a(+)-ESAT6 plasmids were constructed successfully, but no target protein was expressed in pQE30-ESAT6 system. A recombinant protein, about 32kDa, was expressed in pET32a(+)-ESAT6 system. In the presence of 1mM IPTG for 4h, the protein was expressed to the maximum. The protein existed in cytoplasm in soluble form and represented 42% total protein of E.coli. The protein was purified through the Ni-NTA resin and the purity reached 92%. The antigenicity of rESAT6 was confirmed by Western blot in which the sera from the patients with tuberculosis was used.Conclusion: The prokaryotic expression vector pET32a(+)-ESAT6 was constructed successfully, and the purified rESAT6 was obtained. PART Ⅱ CONSTRUCTION AND EXPRESSION OF THE PROKARYOTIC EXPRESSION VECTOR OF MTB lhp GENEObjective: To construct prokaryotic expression vector carrying MTB lhp gene and express it in E.coli, and to purify the rCFP10 with affinity chromatography technique. Methods: The lhp gene fragment with HindIII and BamHI sites was amplified by PCR from MTB genome, then cloned into pQE30 plasmid, sequenced and cloned into pET32a(+) plasmid. The two kinds of prokaryotic expression vectors were transformed into DH5αand BL21(DE3) E.coli respectively, and the lhp gene was expressed in thepresence of IPTG. The rCFP10 was identified by SDS-PAGE and purified with Ni-NTA column. The antigenicity of rCFP10 was confirmed by Western blot.Results: The pQE30-CFP10 and the pET32a(+)-CFP10 plasmids were constructed successfully, but no target protein was expressed in the pQE30-CFP10 system. A recombinant protein, about 32kDa, was expressed in pET32a(+)-CFP10 system. In the presence of 1mM IPTG for 4h, the protein was expressed to the maximum. The protein existed in cytoplasm in soluble form and represented 38% total protein of E.coli. The protein was purified through the Ni-NTA resin and the purity reached 93%. The antigenicity of rCFP10 was confirmed by Western blot in which the sera from the patients with tuberculosis was used.Conclusion: The prokaryotic expression vector pET32a(+)-CFP10 was constructed successfully, and the purified rCFP10 was obtained.PART Ⅲ CONSTRUCTION AND EXPRESSION OF THE PROKARYOTIC EXPRESSION VECTOR OF MTB lhp-esat6 FUSION GENEObjective: To construct lhp-esat6 fusion gene and its prokaryotic expression vector and express it in E.coli and to purify the rCFP10-ESAT6 fusion protein with affinity chromatography technique.Methods: By Gene SOEing technique, a fusion gene fragment with HindIII and BamHI sites was constructed by splicing lhp gene and esat6 gene, then cloned into pQE30 plasmid to construct pQE30-CFP10-ESAT6 plasmid. The recombinant plasmid was transformed into DH5αE.coli, and the lhp-esat6 fusion gene was expressed in the presence of IPTG. TherCFP10-ESAT6 fusion protein was identified...