The Study On The Preparation Of Recombinant Antigens Using E.coli And M.smeg Expression Systems For The Diagnosis Of Tuberculosis

Objective The prokaryotic expression vectors containing the tuberculosis mycobacterium fused genes lhp-esat6 and rdesat-6 were constructed.Meanwhile,the inducible expression of the fusion proteins rCFP10-ESAT6 and rdESAT-6 were performed in the expression systems of E.eoli and M.smegmatis.The fusion proteins were purified using affinity chromatography.The antigenic specificities of the fusion proteins were analyzed by Western-blot.The application of the fusion proteins for the diagonosis of tuberculosis was evaluated.Methods The lhp and esat-6 genes were obtained from Mycobacterium tuberculosis H37Rv genomic DNA by Polymerase Chain Reaction(PCR).The lhp-esat6 fusion gene was constructed with SOE method after the genes lhp and esat-6 were fused in vitro with Linker by three PCR reactions.And then the lhp-esat6 fusion gene was cloned into the plasmids pET28a and pMF41 individually for the construction of the prokaryotic expression vectors pET28a-lhp-esat6 and pMF41-lhp-esat6.In addition,the 2×esat-6 gene was also obtained by PCR from the DNA vaccine HG856A which contains the 2×esat-6 gene.The 2×esat-6 gene was then cloned into the plasmids pET28a and pMF41 for the construction of the prokaryotic expression vectors pET28a-2×esat-6 and pMF41-2×esat-6.The recombinant plasmids pET28a-lhp-esat6 and pET28a-2×esat-6 were transformed into E.coli BL21(DE3) and the fusion proteins rCFP-ESAT6(E.coli) and rdESAT-6 (E.coli) were induced and expressed respectively.Moreover,the recombinatant plasmids pMF41-lhp-esat6 and pMF41-2×esat-6 were electroporated into M.smegmatis(mc²155).The target genes were induced to express the fusion proteins rCFP-ESAT6(M.smeg) and rdESAT-6(M.smeg) respectively with 10%acetamide solution(ultimate density 0.02%).The expression profiles of the above four fusion proteins were identified by SDS-PAGE.And the proteins were purified by Ni²⁺ affinity chromatography column.The antigenic specificities of purified fusion proteins were analyzed by Western-blot with mouse antiserum against rESAT-6.The sensitivity and the specificity of these fusion proteins for the diagonisis of tuberculoiss were assayed by ELISA.The differences of the expression of the recombinant proteins were compared in E.coli and M.smegmatis expression systems with the peripheral blood monocytes as a stimulator.The application of the fusion proteins for the diagnosis of tuberculosis was analyzed as well.Results The prokaryotic expression vectors pET28a-lhp-esat6,pET28a-2×esat-6, pMF41-lhp-esat6 and pMF41-2×esat-6 were constructed successfully and the related fusion proteins were expressed in the form of inclusion body.The fusion proteins were purfied by Ni²⁺ affinity chromatography column and the purity of these proteins were great than 95%by the SDS-PAGE analysis.All the four fusion proteins were antigenic with rESAT-6 mouse antiserum and ELISA.The sensitivity and the specificity of serological diagnosis of tuberculosis with purified rCFP-ESAT6 protein were 25%(5/20) and 95.8%(23/24) respectively;30%(6/20) and 95.8%(23/24) were for the rdESAT-6 protein which were a little bit better than the results of rESAT-6 by literatures.Conclusions The four fusion proteins of tuberculosis mycobacterium were successfully prepared in this study.The fusion proteins were antigenic by Western -blot and ELISA techniques and can be used for detecting humoral immunity in tuberculosis patients.These fusion proteins can be applied for the experiments to detect antibodies or cellular immunity(IGRA) against M.tuberculosis.The rCFP10-ESAT6 and rdESAT-6 fusion proteins may provide the foundations for developing tuberculosis immunological and diagnostic reagents.