The Research On The Serological Diagnosis Value Of Recombinant Protein Of TP47 And TP0453 Antigen Immuno-dominant Epitope Fragment In Infection Of Treponema Pallidum

ObjectiveSyphilis is a very harmful sexually transmitted disease of human over the world that is caused by Treponema pallidum subsp.pallidum(T.pallidum). Enzyme-linked immunosorbent assay(ELISA) based on recombinant antigen has shown some advantages for the clinical laboratory diagnosis of syphilis,since they are easy and quick to perform and economic. ELISA has been used in clinical diagnosis of syphilis extensively. However, the sensitivity and specificity of current testing assays of syphilis are not satisfied. So, it is necessary to optimize traditional antigen and screening other antigens for further specificity and sensitivity of the serological diagnostic kit. In this research, we identified Treponema pallidum specific outer membrane antigens based on bioimformatic analysis, and expressed recombinant protein by genetic engineering technique to provide the basis of theory and practice for the development of quick diagnostic ELISA kit applying to detect T.pallidum infection.Methods1. Treponema pallidum specific outer membrane antigens of TP47 and TP0453 that may having good diagnostic potentiality for T.pallidum infection were selected according to other studies, and the immuno-dominant epitopes of these protein were identified using bioimformatic software DNAstar,and were called TP47'and TP0453'.2. The gene coding TP47'and TP0453'were amplified from T.pallidum complete genome by polymerase chain reaction(PCR) respectively, and the PCR products were cloned into prokaryotic expression vector to generate recombinant plasmid pET22b(+)-TP47'and pQE30-TP0453'.The recombinant vectors which were identified through restriction endonuclease analysis and gene sequencing were transformed and expressed in E.coli BL21(DE3) and E.coli M15 respectively. The expression products were analyzed by SDS-PAGE and purified with Ni2+ affinity chromatograph. Western-blot was used to test the immunoreactivity of TP47'and TP0453'antigens with the sera of patients with syphilis.3. Establishing the double antigen sandwich enzyme linked absorbent assay to detect specific antibody against syphilis in serum of patients. The purified TP47', TP0453'protein and TP17'recombination protein preserved in our department were used to coat the plate respectively,or were pooled in different combination. we have decided the optimal coating combination and dose of recombinant antigen, working concentration of enzyme-labelling antigen ,the adsorbing mode of antigen, blocking buffer and blocking time, serum reacting and coloration time.4. Detecting of clinical serum sample and country quality control serum by using the established ELISA in order to evaluate the methodology.Results1. TP47 and TP0453 were selected as diagnostic antigen for detecting T.pallidum infection, and TP47'(68~410aa) and TP0453'(62~224aa) were estimated as the immuno- dominant epitope of these protein respectively.2. Constructed the high-performance expression vector pET22b(+)-TP47'and pQE30-TP0453'. The results of digestion with restriction DNA enzymes and sequencing of the recombinant plasmids showed the gene was constructed successfully .3. The recombinant plasmid were transformed into E.coli BL21(DE3) and E.coli M15 respectively and induced with IPTG at 37℃. SDS-PAGE analysis showed that the relative molecule mass(Mr) of expressed target protein TP47', TP0453'were 39kDa, 21kDa respectivily ,and the expressing ratio was indicated as 43% and 18% of total bacterial protein by UVP scan. The expression form of TP47'and TP0453'were both inclusion body.4. After purification by Ni2+ affinity chromatograph, we got the target protein TP47'with purity of more than 95% and TP0453'with purity of more than 90%.5. Western blot showed specific reaction of the recombinant protein TP47', TP0453'with T.pallidum IgG positive sera,but no reaction with negatine sera.6. Established and optimized ELISA using different purified antigen. The optimal antigen combinant was TP17'plus TP47', with coating concentration of 4μg/ml and 2μg/ml respectively; enzyme-labelling antigen working concentration of 1:200 and 1:50 respectively; the optimal coating condition is to stay overnight at 4℃; the optimal blocking condition was determined as 1% BSA at 37℃for 2 hours followed by 4℃overnight; the serum reaction condition is 37℃45 min; the coloration time is 15 min at room temperature.7. By using the established ELISA above, we examined the clinical TP serum sample and country quality control serum. The results showed 97.1% for sensitivity, 97.2% for specificity with detecting country quality control serum and the consistency is 97.2%.The minimum detectable quantity is 0.25NCU/ml.Within-run precision is 5.9% and between-run precision is 9.1%. Established ELISA by us has showed 97.7% of sensitivity, 98.7% of specificity . The results of the clinical samples are at equal sensitivity between our method and domestic-made ELISA kit . The specificity is higher than domestic-made ELISA kit.Conclusion1. Estimated the immuno-dominant epitope of TP47', TP0453'protein using bioimformatic software DNAstar.2. Constructed the high-performance expression vector pET22b(+)-TP47', pQE30- TP0453'.3. After purification by Ni2+ affinity chromatograph,we got the target protein TP47'with purity of more than 95%; TP0453'with purity of more than 90%.4. Established and optimized double antigen sandwich enzyme linked absorbent assay using purified TP17', TP47'antigen. The examination results showed that our method had high specificity and sensitivity. The sensitivity between our method and domestic-made ELISA kit are equal in our results of detecting clinical samples and the specificity is higher than domestic-made ELISA kit. So our study provide the basis of theory and practice for the development of quick diagnostic ELISA kit applying to detect T.pallidum infection.