【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。更多还原

【Abstract】 AIM:To construct the eukaryotic plasmid of glucan binding domain(GBD) of Streptococcus mutans 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 immunohistochemistry technique.RESULTS:Restriction endonuclease analysis proved that the recombinant plasmid pVAX1-GbpA/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 VAX1 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.更多还原