Studies On MPT64-Rv1985c Fusion Protein From Mycobacterium Tuberculosis For Diagnosis Of Tuberculosis And Construct A Recombinant BCG Strain

Objective:First part is to construct an expression vector pET32a(+)-MPT64-Rv1985c, express it in E.coli BL21(DE3) and study its diagnostic characteristics by testing sera and pleural effusion samples. The second part is to construct a shuttle plasmid PUV15-MPT64-Rv1985c, transfer it into BCG by electroporation, and verify the protective characteristics of the recombinant protein.Mtheods:The sequences of MPT64and Rv1985c genes were amplified by PCR, and linked by recombinant PCR.The fusion gene was digestted by EcoR I and Hind III, and cloned into pET32a(+) vector. The recombinant protein was expressed under the condition of1mM IPTG,37℃, overnight. The expressed products were renatured and purified by Ni-NTA.The cut-off values were set by adding mean value of OD450to2standard deviations of PPD negative serum and non-tuberculosis pleural effusion samples. The antigen-specific IgG antibody levels against recombinant proteins were evaluated through211serum samples and182pleural effusion samples by E1ISA. The target genes were digested by SphI and NheⅠ, and then cloned into PUV15-GFP shuttle vector.The recombinant vector was electroporated into BCG. The expression of recombinant protein was identified by RT-PCR and Western blot.Results:The recombinant vectors pET32a(+)-MPT64, pET32a(+)-Rv1985c, pET32a(+)-MPT64-Rvl985c were constructed and successfully expressed42kDa,52kDa,76kDa proteins as inclusion bodies in E.coli BL21(DE3). The target proteins were recovered using gradient of urea buffers and then purified by Ni-NAT. Studies on the211serum anti-body IgG revealed that MPT64-Rv1985c recombinant protein achieved sensitivity of50%and specificity of93%. Compared to MPT64and Rv1985c proteins, fusion protein increased the sensitivity by17.1%and14.5%respectively. Antibodies against MPT64-Rvl985c fusion protein achieved sensitivities in detecting pulmonary tuberculosis, extra-pulmonary tuberculosis, active tuberculosis and PPD positive groups were41.9%,55.6%,50%and15.4%respectively, and the specificity was93%. Fusion protein was able to distinguish pulmonary and extra-pulmonary tuberculosis (P=0.014), also discriminate pulmonary tuberculosis and PPD positive group, extra-pulmonary tuberculosis and PPD positive group (P<0.05), suggesting that fusion protein is better than single proteins. On the other hand, we also confirmed that MPT64was able to distinguish PPD positive group and active TB group, but could not diatinguish pulmonary tuberculosis group and extra-pulmonary tuberculosis group, PPD positive group and PPD negative group. Validation of Rv1985c protein in serodiagnosis tests revealed that it was able to distinguish pulmonary tuberculosis group and PPD positive, PPD negative groups, suggesting that Rv1985c was a potential antigen in diagnosis of pulmonary tuberculosis.Studies on the182pleural effusion anti-body IgG revealed that MPT64-Rv1985c fusion protein achieved sensitivities of54.9%in detecting TB pleural effusion and specificities of91.2%respectively in non-TB pleural control group.Compared MPT64protein and Rv1985c protein achieved sensitivities and specificities26.3%,95.6%,48.3%and93.4%respectively, fusion protein could increase the sensitivities in detecting TB pleural effusion for MPT64is28.6%and for Rv1985c is6.6%respectively, but spectificities down by4.4%and2.2%. Statistical analysis revealed that fusion protein can significantly distinguish TB pleural effusion from non-TB pleural control group (P<0.001). The receiver operating characteristic curve of fusion protein revealed that its diagnostic efficency was better than either MPT64protein or Rv1985c protein alone. In order to verify the protective characteristics of fusion protein, we constructed PUV15-MPT64, PUV15-Rvl985c, PUV15-MPT64-Rvl985c recombinant shuttle plasmids, and transferred into BCG by electroporation. Results of RT-PCR and Western blot showed that target proteins expressed in BCG, but no recombinant Rv1985c-BCG was obtained, and the GFP protein about27kDa, the target proteins about51kDa and85kDa were confirmed by Western blot.Conclusion:The recombinant MPT64-Rv1985c protein may be a potential candidate of diagnosis tuberculosis, with it can be increased the humoral response level of single protein.Otherwise, the recombinant MPT64-Rvl985c-BCG was constructed successfully and can express MPT64-Rv1985c fusion gene.