Diagnosis Of Tuberculosis By Biosensor Based On Recombinant TB10.4

Objective In order to provide the technical support for serological diagnosis of tuberculosis,the recombinant proein TB10.4 of Mycobacteria tuberculosis(M.tuberculosis)with good immunological activity was expressed in E.coli BL21 and was used to establish the immunosensor based on gold nanorods(AuNRs)for detection of the antibody to TB10.4 in serums of tuberculosis.Methods The TB10.4 gene was amplified by polymerase chain reaction(PCR)from genomic DNA of M.tuberculosis and was cloned into pMD18-T vector.Positive clones were identified by colony PCR and DNA sequencing.The gene TB10.4 was subcloned into the prokaryotic expression vector pET-28a(+)and identified by PCR and restrictive enzyme digestion with BamH ? and Hind ?.TB10.4 was expressed in E.coli BL21 containing recombinant plasmid pET-TB10.4 when induced by IPTG and was purified by affinity chromatography with His-bindTM column.The immunological activity of TB10.4 was analyzed by western-bolt and the diagnosis efficiency for tuberculosis was evaluated by enzyme-linked immunosorbent assay.The AuNRs were synthesized with seed-mediated method and activated by MUA.EDC and NHS.After that,these AuNRs were connected with the TB10.4 protein to construct the AuNRs immunosensor,and the diagnostic potential of the biosensor for tuberculosis was evaluated.Results TB10.4 gene with the size of about 300 bp was amplified from M.tuberculosis genomic DNA and cloned into pMD18-T vector.The recombinant expression plasmid pET-TB 10.4 was constructed.The recombinant protein TB10.4 expressed in E.coli BL21 was purified by affinity chromatography.The protein was about 10.4 kDa and sepecificly recognized by antibodies in sera of tuberculosis.The sensitivity,specificity,positive predictive value,negative predictive value and the diagnostic efficiency of ELISA using TB10.4 protein as antigen to detect tuberculosis was shown to be 88.5%,97.3%,93.8%,94.8%,94.5%respectively.AuNRs with good dispersity were prepPared with seed-mediated method.TB10.4 was conjugated to AuNRs successfully to develop the biosensor.The sensitivity,specificity,positive predictive value,negative predictive value and the diagnostic efficiency of the AuNRs based on TB10.4 protein to detect tuberculosis was shown to be 96.2%,92.0%,84.8%,98.1%,93.3%.Conclusion Prokaryotic expression plasmid for M.tuberculosis TB10.4 was successfully constructed and the expressed TB10.4 protein has strong immunoreactivity.The AuNRs biosensor using recombinant TB 10.4 as antigen was shown to own a good diagnostic value for tuberculosis.