Molecular Cloning,Expression and Characterization of Cysteine Proteinase Genes in Trichomonas vaginalis

• Main Authors: Lee, Chi-Feng, 李奇峰
• Other Authors: Shaio, Men-Fan
• Format: Others
• Language: zh-TW
• Published: 1997
• Online Access: http://ndltd.ncl.edu.tw/handle/95389585313739004326

碩士 === 國防醫學院 === 病理及寄生蟲學研究所 === 85 === Trichomonas vaginalis is a major pathogen of vaginitis, exocervicitis, and urethritis in women. It has been suggested that T. vaginalis cysteine proteinase (TVCP) may play a role in the pathogenesis of T.vaginalis infection, either acting as a co-factor or a virulence factor. In this study, we used degenerated oligonucleotide primers to amplify TVCP gene fragments by polymerase chain reaction (PCR), Several DNA fragments with expected sizes of about 500 bp were cloned and sequenced. Comparison with the proposed amino acid sequences of other known CPs showed that three TVCP clones were found highly homologous to the conserved region of active domains of CP, and all belong to the cathepsin L/ cathepsin X/ cathepsin O/ papaya proteinase of the papain superfamily. A TVCP fragment with a well-represented sequence was used as a probe for the subsequent experiments. By dot blot hybridization and Southern blot analysis, TVCP recognized several clinical isolates of T. vaginalis; there was no cross-reaction with human DNA or other infectious agents, including Pentatrichomonas horninis and Giardia lamblia. By using Northern blot analysis and reverse transcriptase PCR, expression of mRNA TVCP was demonstrated in different clinical isolates of T.vaginalis. In order to express recombinant TVCP in vitro, TVCP gene was subcloned into the expression vector pQE 30 and expressed in E.coli strain SG13009 [pREP4]. A TVCP fusion protein was purified and demonstrated as a single band with molecular mass (Mr) of 26 kDa in sodium dodecyle sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Rabbit antisera directed against 26 kDa-TVCP fusion protein not only specifically recognize the fusion protein with Mr of 26 kDa, but also detect a product of 30 kDa both in the excretory/ secretory extract and in the lysate of T.vaginalis. The amount of TVCP produced by T.vaginalis was higher at the late logarithmic phase than those at the early logarithmic phase and at the stationary phase during growth. This study suggests that TVCP may play a role in the growth of T. vaginalis. In addition, immunohistochemical study showed that the TVCP were present in cytosol of T.vaginalis. The successful induction of rabbit polyclonal antibodies against 26 kDa-TVCP may provide a useful tool to further investigate the role of TVCP in the immunopathogenesis of T.vaginalis infection.