摘要
目的 构建一含绿荧光蛋白报告基因 (gfp)的防龋基因疫苗 ,利用绿荧光蛋白特有的发光性质 ,示踪防龋基因疫苗在体外的表达情况。方法 采用PCR方法 ,以质粒pPC4 1为模板 ,扩增变形链球菌表面蛋白质抗原(PAC)分子的氨基端A区 (pacA) ,以此作为基因防龋疫苗的目的基因片段 ,与质粒pEGFP_C1重组构建重组质粒pEGFPC1_pacA ,并经酶切、测序鉴定 ;重组质粒瞬时转染COS1细胞后 ,通过荧光显微镜直接观察发绿色荧光的细胞 ,流式细胞仪检测转染细胞的荧光强度 ,RT_PCR扩增pacA基因片段检测重组质粒的体外表达情况。结果 重组质粒中的pacA片段完全来自pac全基因序列 ,pEGFPC1_pacA构建正确 ;荧光显微镜直接观察到了发绿色荧光的细胞 ,重组质粒转染细胞的荧光强度明显高于未转染细胞的荧光强度 ,并证实了重组质粒pacA片段能在哺乳动物细胞中被转录。结论 重组质粒中的绿荧光蛋白基因以及置于gfp基因之后的防龋基因片段均能在体外同时正确表达 。
Objective\ Streptococcus mutans has been proved as a causative bacteria of human dental caries. The surface protein antigen is one of the important pathogenic factors. The A region of the surface protein antigen pac gene (pacA) can enrich T_cells and B_cells epitope. In this study, a DNA vaccine carrying pacA and gfp gene (a reporter gene) for caries prevention was constructed. The DNA vaccine was liable to be traced in vitro and in vivo.Methods\ The fragment of pacA (1 3 kb) was amplified by PCR with the plasmid pPC41 as template, and inserted into a pEGFP_C1 vector. The recombinant plasmid produced was named as pEGFPC1_pacA. After the COS1 cell line was transfected by the recombinant plasmid, the expression of gfp was detected by observing the green fluorescence and measuring the fluorescence intensity, and the expression of pacA was detected by RT_PCR.Results\ Restricted analyzing, sequencing and PCR technique were employed to identify the recombinant plasmid. The phase and orientation of the pacA gene inserted into the vector pEGFPC1 were correct and no changes of their open reading frames were discovered. The transfected COS1 carrying green fluorescent protein (GFP) was observed; the GFP expression level of transfected cells was higher than that of controlled cell. The transcript of pacA gene was confirmed by RT_PCR.Conclusion\ Construction of the recombinant plasmid was successful. The gfp gene and pacA gene in the plasmid was transcribed and expressed simultaneously in the transfected cells. Moreover, detection of GFP is simple, safe and effective for living cells. \;
出处
《华西口腔医学杂志》
CAS
CSCD
北大核心
2003年第3期174-176,共3页
West China Journal of Stomatology
基金
国家自然科学基金 (编号 2 0 0 0 4 0 0 9)
"973"资助项目
关键词
防龋
基因疫苗
体外表达
转染
green fluorescent protein
\ prevention of caries
\ DNA vaccines
\ transfect\
