摘要
目的:构建变异链球菌葡聚糖结合蛋白A葡聚糖结合区的真核表达质粒pVAX1-GbpA/GBD,并观察其在哺乳动物细胞COS-7中的表达。方法:利用基因重组技术构建真核表达质粒pVAX1-Gb-pA/GBD,经酶切分析和测序分析鉴定后,通过脂质体转染法,将其转染至COS-7细胞中,免疫组化SABC法检测其在细胞中的表达。结果:重组质粒pVAX1-GbpA/GBD经EcoR I和BamH I酶切证实携带1.2kb目的基因片段,经测序分析,目的基因正确插入到预先设计的载体位点处。pVAX1-gbpA/GBD转染的细胞胞质呈褐色染色,pVAX1空载体质粒转染的细胞胞质中无着色。结论:成功构建防龋基因疫苗真核表达质粒pVAX1-GbpA/GBD,所携带的基因序列正确,能够在真核细胞COS-7中正确表达目的蛋白。
AIM： To construct the eukaryotic plasmid of glucan binding domain（GBD） of Streptococcus mu- tans glucan binding protein A （GbpA） , and to evaluate the expression of recombinant plasmid pVAX1 -GbpA/GBD in mammalian cells COS-7. METHODS： The eukaryotic plasmid pVAX1-GbpA/GBD was constructed by recombinant DNA technology. The eukaryotic plasmid carrying encoding gene of GBD of Streptococcus mutans GbpA was introduced into COS-7 cells by Lipofectamine. The transient expressed protein in COS-7 cells was detected by immunohisto- chemistry technique. RESULTS： Restriction endonuclease analysis proved that the recombinant plasmid pVAX1-Gb- pA/GBD contained the 1.2kb GbpA/GBD gene. Positive GbpA/GBD expression was detected in plasma of the cells which were transfected with recombinant plasmid pVAX1- GbpA/GBD. The cells which were transfected with VAX! did not show GbpA/GBD expression. CONCLUSION： GpbA/GBD can be translated and expressed in COS-7 cells after being transfected with recombinant plasmid pVAX1-GbpA/GBD.
出处
《牙体牙髓牙周病学杂志》
CAS
北大核心
2010年第6期310-313,340,共5页
Chinese Journal of Conservative Dentistry
基金
贵州省优秀青年科技人才基金[黔科合人字(2005)0509]
贵州省省长基金[黔科教办(2004)07]
贵州省教育厅重点基金资助[黔教科(2004)119]
