Expression, purification and immunocompetence analysis of a Treponema pallidum recombinant protein TP0993
10.3760/cma.j.issn.0412-4030.2013.05.002
- VernacularTitle:梅毒螺旋体TP0993重组蛋白的表达、纯化及免疫活性分析
- Author:
Xiaoping XIE
;
Shuangquan LIU
;
Qiugui ZHANG
;
Yimou WU
- Publication Type:Journal Article
- Keywords:
Treponema pallidum;
Recombinant proteins;
TP0993;
Immunocompetence
- From:
Chinese Journal of Dermatology
2013;(5):305-308
- CountryChina
- Language:Chinese
-
Abstract:
Objective To evaluate the value of a Treponema pallidum (TP) recombinant protein TP0993 in the serodiagnosis of syphilis.Methods A bioinformatics method was used to obtain the sequence of TP0993 gene.The open reading frame (ORF) without upstream non-coding region of TP0993 gene was ligated into the expression vector PET-28a (+),which was then transformed into Escherichia coli Rosetta.Isopropyl-β-d-thiogalactoside (IPTG) was used to induce the expression of TP0993 protein.The expressed protein was purified with nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography.Western blot was performed to evaluate the immunoantigenicity of the protein.New Zealand rabbits were immunized with the recombinant protein for immunogenicity evaluation.Indirect enzyme linked immunosorbent assay (ELISA) was developed by using the purified recombinant protein to coat microwell plates.Anti-TP antibodies were detected by the established ELISA and TP particle agglutination assay (TPPA) in 480 clinical serum samples.Results The prokaryotic expression vector PET-28a (+)-0993 was successfully built,and a fusion protein with a relative molecular weight of about 34 000 Da was attained after IPTG-induced expression and purification.Western blot proved that the recombinant protein could specifically react with clinical sera positive for anti-TP IgG antibodies.Specific humoral response was elicited in New Zealand rabbits by the recombinant protein.Compared with TPPA,the established indirect ELISA showed a sensitivity of 88.3％ and a specificity of 85.8％.There was a consistency of 86.5％ between the indirect ELISA and TPPA.Conclusion The expressed recombinant protein showed favorable immunocompetence,and may serve as a candidate antigen for serodiagnosis of syphilis.
