Diagnosis Of Tuberculosis By Recombinant PstS1 Of Mycobacterium Tuberculosis

Objective: To clone and express the Phosphate-specific transport system protein l(PstS1)of Mycobacterium tuberculosis(MTB),analyze its immunological activity and evaluate its value in the diagnosis of tuberculosis.Methods: The PstS1 gene was amplified by polymerase chain reaction(PCR)from genomic DNA of MTB H37 Rv strain.The target gene was ligated to cloning vector pMD18-T and transformed into the host strain E.coli JM109.Positive clones were identified by colony PCR and DNA sequencing.The PstS1 gene digested with double enzymes was subcloned into the prokaryotic expression vector pET-28a(+).The recombinant expression plasmid pET-28a(+)-PstS1 was extracted from the positive colony and was identified by PCR and double restriction endonucleases.PstS1 was expressed in E.coli BL21 containing recombinant plasmid pET-28a(+)-PstS1 when induced by IPTG.The PstS1 and its solubility was identified by SDS-PAGE and PstS1 was purified by affinity chromatography.Antigenicity of PstS1 was identified by Western-blot.The best concentration of PstS1 and the dilution of patient’s serum for antigen-antibody reaction was confirmed by chessboard titration.ThePstS1 was used to detect tuberculosis by ELISA and its value in the diagnosis of tuberculosis was evaluated.Results: The PstS1 gene with about 1120 bp was amplified by PCR and was identical to the sequence from genomic DNA of MTB H37 Rv strain.The recombinant expression plasmid pET-28a(+)-PstS1 was successfully constructed and PstS1 was obtained when induced by IPTG.PstS1 was purified by affinity chromatography and owned good immunoreactivity when tested by Western-blot.The best reaction concentration of PstS1 was 0.5μg /100μL and the optimal dilution of serum was 1: 100.The sensitivity,specificity,positive predictive value,negative predictive value and diagnostic efficiency of PstS1 protein were 85%,94.9%,94.2%,86.2%,and 89.9% respectively.Conclusion: PstS1 with good immune activities was successfully obtained by recombinant DNA techniques and can be used for tuberculosis detection.