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目的:构建含变异链球菌表面蛋白Spa P的P区编码基因spap-P和葡聚糖结合蛋白Gbp A的葡聚糖结合区编码基因gbd的嵌合体真核表达质粒p VAX1-SPG,并观察其在哺乳动物细胞293T中的表达。方法:通过基因工程技术构建嵌合体真核表达质粒p VAX1-SPG,并采用脂质体转染法将其转染至293T细胞中;然后分别采用免疫组化SABC法和Western-blot检测嵌合蛋白Spa P/P-GBD在真核细胞中的表达。结果:重组真核表达质粒p VAX1-SPG经酶切、测序鉴定证实,其所携带的外源基因片段为2.4 kb的目的基因片段,序列同源性为99%;免疫组化染色结果显示,经p VAX1-SPG转染的293T细胞胞质呈棕褐色染色;Western-blot检测结果显示,分子量为72 k Da的嵌合蛋白Spa P/P-GBD能够被正确表达。结论:构建成功的嵌合体真核表达质粒p VAX1-SPG能在真核细胞293T中正确表达目的蛋白。
OBJECTIVE: To construct a chimeric eukaryotic expression vector p VAX1-SPG containing the gene encoding the dextran-binding domain Gbp of sp-P and dextran-binding protein Gbp A of Streptococcus mutans surface protein Spa P and to observe Its expression in mammalian cells 293T. METHODS: The chimeric eukaryotic expression plasmid p VAX1-SPG was constructed by genetic engineering and transfected into 293T cells by lipofection. Then the expression of chimeric gene was detected by immunohistochemical SABC method and Western-blot Protein Spa P / P-GBD expression in eukaryotic cells. Results: The recombinant eukaryotic expression vector p VAX1-SPG was confirmed by restriction analysis and sequencing. The gene fragment carrying it was 2.4 kb and the sequence homology was 99%. The results of immunohistochemical staining showed that, The cytoplasm of 293T cells transfected with p VAX1-SPG was stained brown; Western-blot showed that the chimeric protein Spa P / P-GBD with a molecular weight of 72 kDa was correctly expressed. Conclusion: The constructed chimeric eukaryotic expression plasmid p VAX1-SPG can correctly express the target protein in eukaryotic 293T cells.