Expression And Purification Of Recombinant Protein MOMP_VD2-VD3 From Chlamydia Pneumoniae And Its Immunocompetence Analysis

Objective:To construct a recombinant vector containing the gene encoding immuno-dominant epitope of major outer membrane protein (Omp) of Chlamydia pneumoniae(Cpn), and to express and purify the recombinant protein, analyzing the immunoreactivity and immunogenicity of recombinant protein, to find a new antigen for the exploiting diagnosis of C.pneumoniae.Methods:The immunodominant epitope of OmpA gene was chosen by EXPASY analysis, then was amplified from C.pneumoniae complete genome by polymerase chain reactions (PCR), The PCR product was directly cloned into pUCm-T vector. After being identified by enzymes cleavage analysis and PCR, both the targeted gene and the prokaryotic vector pET30a were digested with BamH â…  +Hind â…¢ enzymes, and the digested products were linked together by ligase to reconstruct the pET30a-MOMP_VD2-VD3 recombinant plasmid. After constructed the prokaryotic expression system, the recombinant plasmid was induced in E.coli BL21(DE3), and the positive recombinant was identified by enzyme cutting and PCR and sequencing. Recombinant protein MOMP_VD2-VD3 was expressed after induction by IPTG and analyzed by SDS-PAGE and Western blot. MOMP(VD2-VD3) was purified with Ni-NTA-His affinity chromatography and protein concentration was determined by BCA assay. BALB/c mice were immunized with the recombinant protein, antibodies to anti-MOMP_VD2-VD3 in sera were detected with indirected ELISA. The immunoreactivity of the recombinant protein was analyzed by Western-blot and indirected ELISA by using the sera obtained from the patients with Coronary heart Disease.