靶向牙周炎DNA疫苗的构建及体外表达研究

Construction of a targeted DNA vaccine against periodontitis and its expression in vitro

目的:构建以人细胞毒性T淋巴细胞抗原4(Cytotoxic T lymphocyte associated antigen 4,CTLA4)为引导的抗牙龈卟啉单胞菌菌毛蛋白FimA的靶向牙周炎DNA疫苗,并检测其在体外真核细胞中的表达。方法:采用基因重组技术构建携带CTLA4-Ig基因和牙龈卟啉单胞菌fimA基因的重组质粒pCTLA4-FimA,转染COS7细胞,蛋白免疫印记和间接免疫荧光法检测重组质粒的蛋白表达。结果:重组质粒pCTLA4-FimA携带CTLA4-Ig和fimA基因片断。pCTLA4-FimA表达的抗原可与特异性抗FimA抗体发生免疫反应。pCTLA4-FimA转染的COS7细胞中可检测到目的蛋白的表达。结论:成功构建了靶向牙周炎DNA疫苗pCTLA4-FimA,并可在真核细胞中正确表达。

Objective：To construct a CTLA4 targeted DNA vaccine against FimA of Porphyromonas gingivalis and detect its expression in vitro.Method：The CTLA4-Ig gene and fimA gene were amplified from plasmid pGJGLU/GFP and pcDNA3/fimA respectively.The targeted plasmid pCTLA4-FimA was constructed by cloning these two fragments into pCI vector.COS7 cells were transfected with pCTLA4-FimA and the expression of proteins were detected by Western blotting and indirect immunofluorescence.Result：pCTLA4-FimA contains both CTLA4-Ig gene and fimA gene.The expressed protein could react to specific anti-FimA antibody.Green fluorescence was detected in the cytoplasm of pCTLA4-FimA transfected CHO cells incubated with anti-FimA antibody.Conclusion：The targeted DNA vaccine pCTLA4-FimA is constructed successfully and can be expressed correctly in COS7 cells in vitro.