Diagnosis Of Tuberculosis By Recombinant Fusion Protein PstS1-Hsp16.3 Of Mycobacterium Tuberculosis

Objective: To clone and express the recombinant fusion protein Pst S1-Hsp16.3 of Mycobacterium tuberculosis,and establish a nanogold immunosensor and visualized immunosensor based on the recombinant fusion protein Pst S1-Hsp16.3 to test their effiency for the diagnosis of tuberculosis.Methods: Primers were designed according to the pst S1 and hsp16.3gene sequences of the standard Mycobacterium tuberculosis H37 Rv reported in Gen Bank.The gene fragments of pst S1 and hsp16.3 were amplified by PCR.The fusion gene pst S1-hsp16.3 was amplified by overlapping extension PCR and was cloned into p MD18-T vector.After being identified by colony PCR and sequencing,the fused gene fragment was subcloned into prokaryotic expression vector p ET-28a(+).The fusion protein Pst S1-Hsp16.3 was expressed in the E.coli Resseta(DE3)containing plasmid p ET-28a(+)-pst S1-hsp16.3 when induced by Isopropyl ?-D-thiogalactoside(IPTG)and purified by metal chelate chromatography.The immunological activity of Pst S1-Hsp16.3 was analyzed by western-bolt.Gold nanorods were prepared by seed growth method and were chemically modified by 11-Mercaptoundecanoic acid(MUA),1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC),and NHydroxysuccinimide(NHS)in proper order and connected with the fusionprotein Pst S1-Hsp16.3 to construct a Au NRs biosensor.The nanogold immunosensor was used to detect serum antibodies of tuberculosis.Glutathione reduction method was used to prepare gold nanoclusters,which were chemically modified by EDC and NHS,and then linked to goat anti-human Ig G antibodies.Pst S1-Hsp16.3 was used as capture antigen,and gold nanoclusters-goat anti-human Ig G were used as labeled antibodies.A visualized sandwich immunosensor technology was established to detect serum antibodies of tuberculosis,and the diagnostic value of these two methods for tuberculosis was evaluated.Results: The recombinant expression plasmid p ET28a(+)-pst S1-hsp16.3was successfully constructed.After expression and purification,the fusion protein Psts1-Hsp16.3 with a molecular weight of about 60 k Da was obtained and was proved to be immunologically reacted with the antibodies in serums of tuberculosis.A nanogold immunosensor based on the fusion protein Pst S1-Hsp16.3 was constructed,and its sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency were 70.0%,92.0%,89.7%,75.4% and 81.0%respectively.The sensitivity,specificity,positive predictive value,negative predictive value,and diagnostic efficiency of visualized immunosensor constructed based on the properties of gold nanocluster mimicking enzymes were 70.0%,94.0%,92.1%,75.8%,and 82.0%respectively.Conclusion: The fusion protein Pst S1-Hsp16.3 with good immunological activity was obtained by recombinant DNA technology.The nanogold immunosensor and visualized immunosensor based on the fusion protein Pst S1-Hsp16.3 can be used as reference methods for tuberculosis diagnosis.