Diagnosis Of Tuberculosis With Gold Nanorods Biosensor Based On Recombinant Mycobacterium Tuberculosis Protein MPT64

Objective: To construct prokaryotic expression plasmid p ET- 28 a(+)- mpt64 so as to express and purify recombinant protein MPT64. The diagnositic value of recombinant protein MPT64 for tuberculosis was analyzed by Enzyme-linked immunosorbent assay(ELISA) and nanometer gold biosensors, as to provide experimental basis and technical support for serological diagnosis of tuberculosis(TB).Methods: The gene mpt64 was amplified from Mycobacterium tuberculosis standard strain H37 Rv genomic DNA by polymerase chain reaction, and was cloned into p MD18-T vector, positive clones was identified by colony PCR and the sequence was determined by DNA sequencing; The gene mpt64 was subcloned into the prokaryotic expression vector p ET-28a(+), the positive recombinant bacteria was identified by colony PCR and double restrictive enzyme digestion. The recombinant protein MPT64 was abundantly induced to express by IPTG, and was purified by affinity chromatography. The immunological activity of MPT64 was analyzed by western-blot. The concentration of MPT64 was determined by Bradford method. The diagnosis efficiency of recombinant protein MPT64 for tuberculosis was evaluated by ELISA. The gold nanorods(Au NRs) were synthesized with seed-mediated method, after chemical modification with 11- mercaptoundecanoic acid(MUA), 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC) and N-hydroxy-succinimide(NHS), these Au NRs were conjugated with the recombinant protein MPT64 to develop Au NRs biosensor, then the diagnostic potential of the biosensor for TB was detected by seroanalysis.Results:The mpt64 gene was obtained from the Mycobacterium tuberculosis standard strain H37 Rv. The amplified sequence of mpt64 gene was 99% homologous to the sequence of mpt64 gene in Gen Bank detected by sequencing. Prokaryotic expression plasmid p ET-28a(+)-mpt64 was successfully constructed as determined by polymerase chain reaction and enzyme digestion identification. Recombinant protein MPT64 with molecular weight as 26 k Da was successfully expressed when induced by IPTG. The recombinant protein MPT64 was shown with good immunological activity when analyzed by Western-blot. The overall sensitivity, specificity, positive and negative predictive values, and diagnostic efficiency of ELISA which using MPT64 as antigen were 81.4%,94.9%,94.1%,83.6% and 88.1% respectively. After chemical modification, the gold nanorods biosensor based on MPT64 antigen was successfully constructed, and the overall sensitivity, specificity, positive and negative predictive values, and diagnostic efficiency for tuberculosis diagnosis were 91.5%,93.2%,93.1%,91.7%,and 92.4% respectively. Compared ELISA with gold nanorods biosensors for tuberculosis diagnosis by chi square test analysis, the difference between ELISA and gold nanorods biosensors was not statistically significant(P= 0.146>0.05).Conclusion:(1) Recombinant protein MPT64 was successfully expressed in prokaryotic expression system.(2) Recombinant MPT64 antigen was shown to own good immunological activities.(3) Au NRs based on MPT64 antigen was specific and sensitive for the diagnosis of TB.