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背景:葡聚糖结合蛋白B(gbpB)具有良好的免疫原性,但能否作为免疫防龋的候选基因疫苗有待研究。目的:观察gbpB真核表达质粒在哺乳动物细胞COS-7的表达情况。设计:设立对照的实验研究。地点和对象:实验在解放军第四军医大学口腔医学院牙体病科完成,对象为Pbs-gbpB质粒(本实验室构建)。干预:通过基因重组技术构建真核表达质粒pcDNA3.1(+)-gbpB,脂质体转染法将其转染至COS-7细胞。免疫组化SABC法检测,DAB显色。主要观察指标:转染后细胞胞浆、胞核着色情况。结果:pcDNA3.1(+)-gbpB质粒转染的细胞胞浆中呈棕黄色染色,胞核中无着色,pcDAN3.1(+)空载体转染的细胞胞浆及胞核无着色,空白对照组也无着色。结论:质粒pcDNA3.1(+)-gbpB转染COS-7后能够在细胞内翻译、表达,表达的蛋白质位于胞浆中,可与抗GbpB抗体特异性结合,具有抗原性,可作为基因疫苗。
BACKGROUND: Dextran-binding protein B (gbpB) has good immunogenicity. However, whether it can be used as a candidate gene vaccine for immune and caries prevention remains to be studied. Objective: To observe the expression of gbpB eukaryotic expression plasmid in mammalian cells COS-7. Design: Set up a controlled experimental study. Location and Subjects: The experiment was performed in the Department of Dentistry, Fourth Military Medical University of Stomatology and targeted at the Pbs-gbpB plasmid (constructed in our laboratory). Intervention: Eukaryotic expression plasmid pcDNA3.1 (+) - gbpB was constructed by gene recombination technology and transfected into COS-7 cells by lipofection method. Immunohistochemical SABC assay, DAB color. MAIN OUTCOME MEASURES: Cytosolic and nuclear staining after transfection. Results: The cytoplasm of pcDNA3.1 (+) - gbpB plasmid transfected cells showed brownish yellow staining and no staining in the nucleus. The cytoplasm and nucleus of pcDAN3.1 (+) empty vector transfected cells had no staining and blank The control group also had no coloring. CONCLUSION: The plasmid pcDNA3.1 (+) - gbpB can be translated and expressed in cells after transfection with COS-7. The expressed protein is localized in the cytoplasm and specifically binds to anti-GbpB antibody. It is antigenic and can be used as a gene vaccine .