Expression And Preliminary Application In Clinical Detection Of Treponema Pallidum Hypothetical Protein Tp0693

Objective:To construct the prokaryotic expression vector of the Treponema pallidum hypothetical protein Tp0693, and induce its expression in E. coli. Evaluate its clinical diagnosis value in syphilis serology, and provide experimental evidence for the discovery of new Treponema pallidum candidate antigens.Methods:Obtain the Tp0693 gene sequence from Genbank, and design upstream and downstream double primers. We use Tp Nichols standard strain DNA as a template to amplify Tp0693 gene,then construct the expression vector pET30a-Tp0693 and induce expression in E.coli BL21(DE3) bacteria. After protein is purified and coated in microtiter plates, we establish ELISA method to detect its immunoreactivity with the syphilis serum(201 cases), cross-serum(66 cases) and negative serum(100 cases). Meanwhile,we compare the Tp0693-ELISA with the TPPA, RPR assay results by statistical analysis. We can preliminary evaluate the Tp0693-ELISA value in the diagnosis of syphilis.Results:(1) Amplified fragment was consistent with the length of the target gene. And the gene fragment inserted into recombinant plasmid by double digestion and gene sequencing was proved to be Tp0693 gene.(2) SDS-PAGE results showed that the relative molecular weight of the aim protein Tp0693 was about 50 kDa, and WB results showed that the target protein had specific immune-reactivity with Anti-His monoantibody and syphilis positive serum.(3) The S/CO values from Tp0693-ELISA was grouped from low to high. Found that the positive rate of TPPA and RPR increased with S/CO value increases. When the S/CO?3.001, the positive rates of TPPA test were 100%, and the RPR test were 79.56%, performed with SPSS17.0 X2 trend test, P <0.01.(4) With S/CO as the detection variable quantity and TPPA as the gold standard to draw ROC curve. Area under the curve is 0.990, indicating good diagnostic results.(5) With S/CO value 2.721 as the critical point, the specificity of Tp0693-ELISA is 100%, the sensitivity is 91%. The Tp0693-ELISA is consiste with the TPPA, and higher than the RPR sensitivity.(6) With SPSS17.0 to analyze the S/CO value amongTp0693-ELISA assay with TPPA, RPR titer detected correlation in the syphilis positive samples. the correlation coefficient with TPPA titer is 0.116, P> 0.05, with RPR titer is 0.176, P> 0.05, which show the syphilis positive samples Tp0693-ELISA S / CO value is not correlated with TPPA, RPR titers.(7)With the S/CO ranging from 1.368 to 2.721 as Tp0693-ELISA method detecting gray zone, a higher proportion was found in the latent syphilis 14.8%(16/108); cross-serum samples in the gray zone typhoid fever and rheumatism higher proportion were 80%(8/10) and 55.2%(16/29). TPPA, RPR titers scattered from high to low in the gray zone.Conclusions:The constructed PET30a-Tp0693 expression vector was successfully expressed in E.coli BL21, and the established Tp0693-ELISA was of highly specificity, good sensitivity and expected to become a Tp candidate diagnostic antigen.