Expression, purification and immunocompetence analysis of a recombinant membrane protein, Tp0136, of Treponema pallidum / 中华皮肤科杂志

ObjectiveTo clone and express the Tp0136 gene of Treponema pallidum,to purify the recombinant protein and to evaluate its immunocompetence.MethodsThe full-length Tp0136 gene was synthesized,then subcloned into the expression vector pET28a to construct a recombinant plasmid,pET28a-Tp0136,which was subsequently transfected into E.coli Rosetta for protein expression.The recombinant protein was purified with nickel-nitrilotriacetic acid(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 Tp0136 (rTp0136)protein,and anti-Tp0136 polyclonal antibodies in sera of the rabbits were examined by indirect enzyme linked immunosorbent assay(ELISA) with rTp0136 protein as the coating antigen.Also,positive sera were obtained from patients with syphilis and examined by Western blot to identify the immunoreactivity of the rTp0136 protein.ResultsThe E.coli expression vector pET28a-Tp0136 was constructed successfully,and rTp0136 protein was also successfully expressed with a molecular weight of about 50 kD and a purity above 95％.High titres of anti-rTp0136 antibodies were detected in sera of rabbits immunized with the rTp0136 protein,and Western blot showed that the rTp0136 could specifically react with the sera from syphilitic patients,which proved the high immunogenicity and immunoreactivity of the recombinant protein.ConclusionsThe full-length Tp0136 membrane protein is successfully expressed with a high immunocompetence,and Tp0136 membrane protein may play an important role in the pathogenic mechanisms of Treponema pallidum.

Soluble expression, purification and characterization of recombinant Tp0136 selective fragment from Treponema pallidum / 中华微生物学和免疫学杂志

Objective To express and purify recombinant Tp0136 epitope fragment, and study the immunity activity. Methods The Tp0136 selective fragment(Tp0136B) gene was devised by the surface property analysis, solvent-accessible suface calculateions, secondary structure function region analysis, and was inserted between the sites of Nde Ⅰ and Not Ⅰ in pET22b ( + ) . The recombinant plasmid was expressed in E. coli BI21. After nickel ion metal affinity chromatography, the antigenic and immune reactivity of rTp0136B was confirmed. Then indirect ELISA with the rTp0136B as coating antigen was performed to detect the anti-Tp0136 antibody in sera from 100 normal human controls and 131 primary syphilis patients. Results The rTp0136B was soluble expressed with a molecular weight of about 28 000 and was obtained with a purity of ＞98% by chromatography. Western blot proved that the rTp0136B could specifically react with anti-Tp0136 polyclonal antibody. Specific humoral response was elicited by the recombinant protein in Japan negative. The positive detection rate in sera from primary syphilis patients was 85.5%. Conclusion This result suggested that the recombinant Tp0136 epitope fragments have a satisfactory immunocompetence,which may have applications in the serodiagnosis of primary syphilis.