Expession of Tp0319 recombinant protein from Treponema pallidum and analysis of its immunocompetence
10.3760/cma.j.issn.0412-4030.2010.05.012
- VernacularTitle:梅毒螺旋体Tp0319重组蛋白的表达及免疫活性研究
- Author:
Shuangquan LIU
;
Shiping WANG
;
Yongjian XIAO
;
Yimou WU
;
Feijun ZHAO
;
Tiebing ZENG
;
Yuejun ZHANG
;
Dongmei GAO
- Publication Type:Journal Article
- Keywords:
Treponema pallidum;
Recombinant proteins;
Immunocompetence
- From:
Chinese Journal of Dermatology
2010;43(5):332-335
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone, express Tp0319 gene from Treponemapallidum (T. pallidum), and to assess the immunocompetence of recombinant protein. Methods The immuno-dominant region of Tp0319gene was chosen by computer analysis, amplified from T. pallidum complete genome by PCR, subcloned into the expression vector pQE32 to construct a recombinant plasmid, pQE32/Tp0319, which was then expressed in E. coli M15. The recombinant protein was purified with Ni-NTA affinity chromatography, and identified by using sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and Western blot. New Zealand rabbits were immunized with the recombinant protein, and the titer of anti-Tp0319 antibodies in sera from immunized rabbits were measured with indirect ELISA. Also, indirect ELISA with the recombinant Tp0319 as coating antigen was performed to detect the anti-Tp0319 antibody in sera from 200 normal human controls and 200 patients with syphilis. Results The prokaryotic expression vector pQE32/Tp0319 was constructed successfully, and the recombinant protein Tp0319 with a molecular weight of about 30 000 was attained. Specific humoral response was elicited by the recombinant protein in New Zealand rabbits and the specific antibody titer was more than 1: 10 240 after immunization for 3 times. Western blot proved that the recombinant protein could specifically react with anti-T. pallidum IgG antibody-positive sera. Indirect ELISA was successfully developed with the recombinant Tp0319, and detected antibodies to T. pallidum in control sera with a sensitivity and specificity of 100% (40/40), respectively. Compared with T. pallidum particle agglutination (TPPA) assay, the sensitivity and specificity of the indirect ELISA were 92.6% and 100%, respectively, in the detection of T. pallidum in sera from patients and controls, and the concordance between the indirect ELISA and TPPA was 96%. Conclusions The prepared recombinant protein shows a satisfactory immunocompetence, which may lay a foundation for its further application in the serodiagnosis of syphilis.
