Construction, Expression And Identification Of Chlamydia Trachomatis Major Outer Memebrane Protein Encoding Recombinant Plasmid

Chlamydia trachomatis(Ct) is a human pathogen which causes trachoma, non-gonococcal urethritis and many other diseases of Genitourinary tract.18 serotypes of Ct have Major Outer Membrane Protein(MOMP) which not only plays important role for adhesion of Chlamydia trachomatis to host cells,but also itself can be important immunogen to stimulate the host to generate cellular and humoral immune responses. ompA gene is the structure gene for coding MOMP,so to prepare strong immunogenic MOMP antigen according to the specificity of ompA gene has fundamental meaning for the research of pathogenesis,and development of diagnostic kit,peptide chip,and vaccine of Ct.In the present study,we anaylsed the amino acid and base sequence of ompA gene from 18 serotypes of Ct,and found that B type ompA gene differs from other types with 2-3 amino acids.So we decided to choose the epitope of B/TW-5 ompA which is 62 amino acids long.ompA gene was synthesized by Invitrogen and was recombined with pET-41a and transformed into E.coli BL21(DE3).The sequencing of recombinant plasmid confirmed that the ompA gene we had cloned matched the optimized sequence. Expression of the recombinant plasmid was induced by IPTG.The protein was analyzed by SDS-PAGE and purified by Ni-IDA.The expressed fusion protein was about 34KD and the proper condition for purification is as follows,binding buffer:Na₂HPO₄: NaH₂PO₄ 3:2(20mmol/L ),8M/L urea,5mmol/L imidazole,pH8.0;Elution buffer: Na₂H PO₄:NaH₂PO₄ 3:2(20mmol/L),8M/L urea,500mmol/L imidazole, pH8.0,eluted with 5%,10%,15%,20%,25%gradient.Antibody-binding activity of purified MOMP protein was confirmed by ELISA in the clinical serum specimen.The sensitivity is 85.7%,and the specificity is 87.8%.