Expression Of Recombinant Constructed CopN Gene Of TTSS From Standard Strain Of Chlamydia Trachomatis Serovar E And Its Protein Identification

Chlamydiae are a worldwide major cause of sexually transmitteddiseases and infectious blindness, and they cause respiratory tractinfections and are associated with atherosclerosis; but now theirbasic pathogenicity mechanisms remain elusive. TypeⅢsecretionsystem(Type three secretion system, TTSS) is independent of thesecretion system. Various gram-negative animal and plant pathogensuse TTSS as a basic virulence mechanism[1]. Secretion of bacterialpathogenicity proteins by the typeⅢpathway and their injection intothe cytosol of animal or plant cells. Redirection of cellular signaltransduction may result in disarmament of host immune responses orin cytoskeletal reorganization, establishing subcellular niches forbacterial colonization.. Chlamydia as obligate intracellular bacteriaalso has TTSS. The CopN (Chlamydia outer protein N) protein is theimportant part of Chlamydial TTSS and it is predicted to modulate theprotein secretion of chlamydial TTSS. The research of the CopNprotein may clarify the mechanism of TTSS. Then the pathogenesisof the Chlamydia may be illustrated.Object:To construct the prokaryotic expression recombinant of copN-pTrcHisA and to detect the CopN protein of the recombinantconstructed copN gene using the methods of SDS-PAGE andWestern Blotting which would further facilitate the research on theexpression and function of the CopN protein.Method:The plasmid-pTrcHisA was choosen as the vector and copN genewhich encodes one modulator protein of TTSS of chlamydiatrachomatis (C.t) as the target DNA. Cell culture of standard strainsof E serotype was used. After lysised, samples were used as template for polymerase chain reaction(PCR). Firstly, plasmid primerof C.t was used to identify the existence of C.t. Secondly, throughpolymerase chain reaction, specific copN gene was amplified usingits specific primer. Thirdly, used two different restriction endonucleasetreating the target gene and plasmid vector. Then inserted DNAfragment into plasmid vector and transformed the recombinant intoE.coli. And at last, screened positive recombinant, including culturingthe E.coli on LB agarose plate containing ampicillin, amplifing copNgene from recombinant through the PCR method, and digestingrecombinant by restriction endonuclease. Finally, the inserted DNAfragment was sequenced by sending the positive recombinant to thesequence corporation.Then for the immunodetection, we did the SDS-PAGE, for which thebacteria was grown in the presence of IPTG.Then western blottingwas performed using two antibodies the 1^st—anti- His-Tag antibodyand the 2^nd-antibody—peroxidase anti mouse IgG (H+L). By westernblotting, we got the band in the nitrocellulose paper which proves thedetection of CopN.Result:The prokaryotic expression recombinant copN-pTrcHisA wasconstructed successfully. The inserted copN gene has the samesequence with copN gene of C.t. provided by the Genebank. And theSDS-PAGE and Western Blotting proved the expression andidentification of the CopN protein.Conclusion:Above results manifests that the test has successfully expressed therecombinant of CopN gene from chlamydia trachomatis standardstrain of E serotype and had identified its protein.