[Construction of the prokaryotic expression vector of MTB lhp gene and its expression]

To construct prokaryotic expression vector carrying lhp gene and express it in E.coli.

The MTB lhp gene was amplified by PCR and then cloned into plasmid pQE30. After sequencing, the gene was cloned into plasmid pET32a(+) to construct recombinant prokaryotic expression vectors pQE30-CFP10 and pET32a(+)-CFP10.

After transformation of the E.coli and induction with 1 mmol/L of IPTG, no additional protein was expressed in pQE30-CFP10 system, but recombinant target protein with M(r) 20 000 or so was expressed in pET32a(+)-CFP10 system, and the expressed protein was maximum when induced with IPTG for 4 h. The expressed protein existed in cytoplasm in soluble form and amounted to 38% of total protein of E.coli. Western blot analysis showed that the protein had good antigenicity. The purity of the protein purified through the Ni-NTA resin reached 93%.

The prokaryotic expression vector pET32a(+)-CFP10 was constructed successfully and the rCFP10 protein was obtained, which laid the foundation for application of the rCFP10.