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[Cloning, expression and immunogenicity analysis of cysteine proteinase 3 of Trichomonas vaginalis]
Wan-Zhong Jia1, Zhi Li, Liang Zhao
1Center for Parasitic Organisms, State Key Laboratory of Biocontrol, School of Life Sciences, Zhongshang University, Guangzhou 510275, China.
Objective:
To study the humoral antibody response of mice to the recombinant Trichomonas vaginalis cysteine proteinase (TvCP3) in order to investigate the function of the proteinase and its application in diagnosis.
Methods:
T. vaginalis cysteine proteinase gene 3 (TvCP3) was cloned by using PCR, and was inserted into the expression vector pET28b. The recombinant plasmid pET28b-TvCP3 or pET28b-TvCP3C (a matured and pre-matured enzyme fragment) was then transformed to Escherichia coli BL21(DE3). The recombinant protein was expressed in E. coli, purified by IMAC (immobilized metal affinity chromatography), and was used to immune BALB/c mice. The mice were divided into groups TvCP3, TvCP3C and control, 6 in each group. The first and second injections for each mouse were administered with 25 microg purified TvCPs which was emulsified with an equal volume of Freund's complete and incomplete adjuvants, respectively. Two more injections were done using 12.5 microg purified antigens without adjuvants, with 2 weeks interval between the first three injections and one week interval between the 3rd and 4th injections. The murine serum samples were detected one week post the 4th injection. The specific IgG antibody in the serum against the recombinant protein was evaluated by ELISA and Western blotting.
Results:
The expression level of TvCP3 and TvCP3C reached to more than 25% of the total amount of proteins expressed by the bacteria respectively, and the purity in both of them was more than 80% after purified by cobalt-based IMAC resin. ELISA showed that both purified recombinant TvCP3 and TvCP3C induced a high titer of antibodies in the immunized mice (1/204,800 and 1/102,400, respectively). Western blotting analysis indicated that the antibodies reacted with a specific band of the whole T. vaginalis antigens or soluble fractions from T. vaginalis cultures.
Conclusion:
The recombinant TvCP3 and TvCP3C proteins have been highly expressed in E. coli BL21 (DE3) and the expressed products can induce high level of antibodies in BALB/c mice, which recognized a specific band of proteins from T. vaginalis soluble fractions and cultures in vitro.
