Diagnosis Of Tuberculosis With Gold Nanoparticles Biosensor Based On The Recombinant Fusion Protein TB10.4-Hsp16.3 Of Mycobacteria Tuberculosis

Objective:To clone and express the recombinant fusion protein TB10.4-Hsp16.3 of Mycobacteria tuberculosis(M. tuberculosis) and analyze the value of diagnosis of tuberculosis,then establish the Gold Nano-Biosensor (AuNRs) based on recombinant fusion protein TB10.4-Hsp16.3 to detect tuberculosis in order to provide new experimental basis and technical support for serological diagnosis of tuberculosis. Methods:(1) The primers of TB10.4 and Hsp16.3 of M. tuberculosis were designed and synthesized based on their sequences in GenBank; genes of TB 10.4 and Hsp16.3 were amplified by polymerase chain reaction (PCR) from M. tuberculosis H37Rv genomic DNA respectively, the fusion gene TB10.4-Hsp16.3 was amplified by overlap PCR and was cloned into pMD18-T vector. Positive clones were identified by colony PCR and DNA sequencing. The fusion gene TB10.4-Hsp16.3 was subcloned into the prokaryotic expression vector pET-28a(+) and identified by PCR and double enzyme digestion. (2) Recombinant fusion protein TB10.4-Hsp16.3 was expressed in E.coli BL21 containing recombinant plasmid pET-TB10.4-Hsp16.3 when induced by isopropyl (3-D-thiogalactoside (IPTG); Bacterial precipitation was dissociated by ultrasonic lysis method, and the inclusion body was dissolved in 8mol/L urea and the fusion protein TB10.4-Hsp 16.3 was renatured by low urea gradient and then purified by nickel chelate chrom- atography.The immunological activity of TB10.4-Hsp16.3 was analyzed by western-bolt and the diagnosis efficiency for tuberculosis was evaluated by enzyme-linked immunosorbent assay. (3) The gold nanorods (AuNRs) were synthesized with seed-mediated method. After being chemically modified with MUA, EDC, and NHS, these AuNRs were connected with the fusion protein TB10.4-Hsp16.3 to construct the AuNRs biosensor,and the diagnostic potential of the biosensor for tuberculosis was evaluated. Results:(1) The amplified fusion gene TB10.4-Hsp16.3 was about 750bp;the recombinant expression plasmid pET-TB10.4-Hsp16.3 was constructed through subcloning the fusion gene TB10.4-Hsp16.3 into prokaryotic expression vector pET-28a(+). (2) The fusion protein was expressed in E.coli BL21 containing plasmid pET-TB10.4-Hspl6.3 when induced by IPTG and was purified from the lysate; the fusion protein was about 29 kDa and recognized by antibodies of tuberculosis sera. The sensitivity, specificity, positive predictive value, negative predictive value and the diagnostic efficiency of ELISA using fusion protein TB10.4-Hsp16.3 as antigen to detect tuberculosis was showed to be 89.3%,90.7%,90.9%,89.1%,90% respectively. (3) The AuNRs biosensor based on the fusion protein TB10.4-Hsp16.3 was constructed and the sensitivity, specificity, positive predictive value, negative predictive value and diagnosis efficiency for tuberculosis was 83.9%,85.2%,85.5%,83.6% and 84.5% respectively.Conclusions:(1)The recombinant fusion protein TB10.4-Hsp16.3 was obtained by recombinant DNA technology and purified by affinity chromatography. (2) The recombinant fusion protein TB10.4-Hsp16.3was showed to own good immunological activities and can be used to test tuberculosis. (3) The AuNRs biosensor based on the fusion protein TB10.4-Hsp16.3 is useful for the diagnosis of tuberculosis.