[Preparation, characterization and preliminary application of recombinant protein AP33 of trichomonas vaginalis]
Shao-hui Liang1, Hui-cong Huang, Chang-wang Pan
1Department of Parasitology, Wenzhou Medical College, Wenzhou 325035, China.
Objective:
To clone ap33 gene of Trichomonas vaginalis( T. v), construct prokaryotic expression system of the gene and identify its antigenicity and immunogenicity.
Methods:
The total RNA was extracted from a clinical isolate Tv317 and the cDNA was synthesized by reverse transcription. The ap33 gene from cDNA of Tv317 was amplified by PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a (+) with inserted ap33 gene was constructed. Recombinant fusion protein AP33 was expressed in E. coli strain BL21DE3 induced by IPTG at different dosages. Western blotting was applied to determine immunoreactivity of the recombinant fusion protein AP33 with antibody against whole cell of T. v. Double agar diffusion was applied to determine immunogenicity of the recombinant fusion protein AP33 with rabbit antiserum immunized with the recombinant fusion protein AP33, and ELISA with antigen of T. v whole cell was applied to determine immunogenicity of the recombinant protein AP33. Positive human sera were tested by ELISA with the recombinant fusion protein AP33.
Results:
High homology of nucleotide and amino acid sequences was revealed between the cloned ap33 and the corresponding gene. The recombinant protein showed a high expression level. The recombinant protein was recognized by anti-T. v polyclonal antibody from rabbit, and showed a high titer. The clinical T. v isolates showed high ap33 expression level and stimulated the production of specific antibody. Antibody against AP33 was detected in 78% of the 50 patients infected with T. v by ELISA.
Conclusion:
A prokaryotic expression system of T. v ap33 gene has been established. The expressed fusion protein AP33 shows satisfactory antigenicity and immunogenicity.
