Expression And Characteristic Of CLPC2Protein Of Mycobacterium Tuberculosis

ObjectiveTo construct the prokaryotic expression plasmid for ClpC2of MTB,and express in E.coli BL21.The rClpC2was purified by affinitychromatography. The rabbits were immunized with purified rClpC2andrabbit antiserum against ClpC2(anti-ClpC2) was prepared by hypodermicimmunization. The rClpC2and anti-ClpC2were taken as antigen orantibody to detect healthy and pulmonary tuberculosis patients, thespecificity and sensitivity were measured. It provides an experimental basisfor potential application of rClpC2in the diagnosis of tuberculosis. Toobserve the growth situation of E.coli BL21with the overexpression ofrecombinant protein when it under every different stress condition. Thisexperiment laid a foundation for further study on biological function ofClpC2protein of MTB.Methods1. The ClpC2gene was amplified by PCR from the genome of MTBH37Rv strain, and cloned into pET32a(+) plasmid to construct recombinant plasmid pET32a(+)/ClpC2. The constructed recombinant plasmid wastransformed into E.coli BL21and expressed with IPTG induction. TherClpC2was analyzed by SDS-PAGE and Western-blot.2. The rClpC2was purified by affinity chromatography. The rabbitswere immunized with purified rClpC2protein mixed with Freund’ adjuvant,the anti-rClpC2was prepared by hypodermic immunization. The serumwas collected at the7thdays after the fifth immunization. The specificity ofanti-rClpC2was detected by Western-blot. The titer of anti-rClpC2wasdetected by double immunodiffusion and indirect ELISA. We took thepurified rClpC2protein as diagnostic antigen to detect pulmonarytuberculosis patients (n=50). We also used anti-rClpC2polyclonal antibodyas coating antibody to detect specific antigen in the same serum samples.The specificity and sensitivity were measured.3. To observe the growth curve of E.coli BL21with theoverexpression or coexpression of recombinant protein when it under thecondition of oxidative stress, acid stress, alkali stress and ethanol stress.The impact of the overexpression of recombinant protein on E.coli BL21biofilm formation was detected by Crystal violet semi-quantitative method.Results1. After identified by DNA sequencing and restrictive digestion, therecombinant plasmid pET32a(+)/ClpC2was constructed successfully. Theexpressed recombinant protein about46000was expressed in E.coli BL21. rClpC2can bind with the antibody in the serum of the mouse immunized byMTB.2. The purity of rClpC2was88.5%by Bandscan analysis. Thespecificity of the anti-rClpC2was determined with Western-blot. The titer ofanti-rClpC2was1:32and1:320000by double immunodiffusion and ELISAdetected respectively. We took the purified rClpC2protein as diagnosticantigen to detect pulmonary tuberculosis patients (n=50). We also usedanti-rClpC2polyclonal antibody as coating antibody to detect specificantigen in the same serum samples by ELISA. The results show that thesensitivity was46%and40%respectively, specificity was90%respectively.3. The overexpression of ClpP1was conducive to E.coli BL21againstoxidative stress, the coexpression of ClpP1and ClpC2was conducive tothe growth and against acid stress and alkali stress of E.coli BL21. Theoverexpression of rClpC2was not contribution to biofilm formation ofE.coli BL21.Conclusions1. The prokaryotic expression recombinant pET32a(+)/ClpC2wassuccessfully constructed,and ClpC2protein was expressed in E.coli BL21.2. The anti-rClpC2polyclonal antibody was prepared successfully,which may be applied to the preliminary diagnosis of MTB infection.3. The ClpP1protein was related to oxidative resistance of E.coli BL21, ClpP1and ClpC2protein were related to acid resistance and alkaliresistance of E.coli BL21.