重组HBsAg-GFP载体的构建及其在293T细胞中的表达

发布时间：2018-03-08 13:15

本文选题：乙肝表面抗原　切入点：真核表达　出处：《实用医学杂志》2013年20期 　论文类型：期刊论文

【摘要】：目的:构建pCMV-HBsAg-GFP真核表达载体,鉴定重组蛋白在293T细胞中的表达及抗原性。方法:以HBV转基因鼠C57BL/6J-TgN(AlblHBV)44Bri基因组DNA为模板扩增HBsAg基因片段,并将其插入到带绿色荧光蛋白标签的真核表达载体pCMV-AC-GFP。通过阳离子聚合物将重组质粒pCMV-HBsAg-GFP转入293T细胞,荧光显微镜观察重组蛋白的表达,ELISA和Western Blotting鉴定其抗原性。结果:1%琼脂糖凝胶电泳结果显示,PCR扩增片段大小符合HBsAg基因699 bp的理论值。重组质粒经PCR、酶切鉴定后也与理论值相符。荧光显微镜显示,转染293T细胞48 h后,约50%呈亮绿色,细胞形态好,荧光表达强度高。ELISA和Western Blotting鉴定结果均为阳性。结论:成功构建了pCMV-HBsAg-GFP真核表达质粒,其表达的重组蛋白与天然HBsAg具有相同的抗原性。
[Abstract]:Objective: to construct pCMV-HBsAg-GFP eukaryotic expression vector and identify the expression and antigenicity of the recombinant protein in 293T cells. Methods: the HBsAg gene fragment was amplified by using the genomic DNA of C57BL / 6J-TgNfAlBHBV V44Bri transgenic mice as template. It was inserted into the eukaryotic expression vector pCMV-AC-GFPwith green fluorescent protein tag. The recombinant plasmid pCMV-HBsAg-GFP was transferred into 293T cells by cationic polymer. Fluorescence microscopy was used to observe the expression of the recombinant protein. Elisa and Western Blotting were used to identify its antigenicity. Results the results of 1% agarose gel electrophoresis showed that the amplified fragment size of the recombinant plasmid was in accordance with the theoretical value of 699bp of HBsAg gene. The fluorescence microscope shows that, After transfection of 293T cells for 48 h, about 50% cells showed bright green, good morphology, high intensity of fluorescence expression. Elisa and Western Blotting were positive. Conclusion: the eukaryotic expression plasmid of pCMV-HBsAg-GFP was constructed successfully. The expressed recombinant protein has the same antigenicity as natural HBsAg.
【作者单位】：南方医科大学南方医院儿科;南方医科大学基础医学院免疫学教研室;
【基金】：国家自然科学基金资助项目(编号:81201295)
【分类号】：R392.1

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