文档介绍：pSG5/NS5A5BΔC质粒的构建及在Huh7细胞中的表达
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作者:王雪萍, 李富军, 邓琳, 长野基子, 北山喜久美, 堀田博
【摘要】目的: 构建pSG5/NS5A5B△C质粒, 并检验其在Huh7细胞中的瞬时表达。方法: 使用已经构建的pTM1NS2NS5A5B△C质粒为模板, 根据pTM1、 HCV NS5A5B△C、 pSG5序列和酶切位点特点设计引物, PCR扩增得到HCV NS5A5B△C基因片段, 将目的片段插入pSG5载体中, 经筛选阳性克隆、 SacⅠ和BglⅡ分别酶切鉴定后, 用FuGene 6转染试剂转染入Huh7细胞。用免疫荧光染色、 Western blot检测HCV NS5A5B△C在Huh7细胞中的表达。结果: 限制性内切酶SacⅠ和BglⅡ分别酶切鉴定后, 琼脂糖凝胶电泳结果显示酶切片段大小和插入方向均与预计一致。荧光显微镜下可见转染的Huh7细胞表达带有绿色荧光的HCV NS5A5B△C蛋白, 该蛋白主要表达于细胞质, 表达率达40%以上。Western blot结果也证实在转染pSG5/NS5A5B△C的Huh7细胞中出现了大小与HCV NS5A5B△C (82 ku)一致的条带。结论: 成功构建了pSG5/NS5A5B△
C质粒, 并在Huh7细胞中成功瞬时表达, 为进一步研究HCV蛋白的功能奠定了基础。
【关键词】丙型肝炎病毒; 非结构蛋白5A5B; pSG5; Huh7细胞
[Abstract] AIM: To construct the plasmid pSG5/NS5A5B△C, and to investigate its expression in Huh7 cells. METHODS: The plasmid pTM1NS2NS5A5B△C was taken as the template. The primers were designed according to the characteristics of the sequence and endoenzyme cleavage sites in HCV NS5A5B△C and vector pTM1, pSG5. The HCV NS5A5B△C gene fragment was amplified by PCR from pTM1NS2NS5A5B△C and inserted into vector pSG5. The positive clones were screened by Ampicillin and identified by the restriction endoenzyme SacⅠand BglⅡ digestion and agarose gel electrophoresis. The constructs were transfected into Huh7 cells with FuGene 6 reagents. Immunofluorescence and Western blot were performed to detect the expression of the constructs in Huh7 cells. RESULTS: Endoenzyme digestion analysis showed that the size and the inserting orientation of the fragment met the design expectation. Immunofluorescence staining displayed the expression of HCV NS5A5B△C protein, which was located in the cytoplasm, and the expression rate reached as high as 40%. SDS
PAGE analysis showed that the relative molecular mass of the expressed product by pSG5/NS5A5B△C was about 82 ku, which was consistent with the theoretical value. CONCLUSION: pSG5/NS5A5B△C is essfully constructed, and it can be expressed transiently in Huh7 cells, which wou