p55PIK慢病毒表达载体的构建及鉴定

发布时间：2018-06-26 00:43

  本文选题：慢病毒 + p55PIK　； 参考：《华中科技大学》2011年硕士论文

【摘要】：目的: 构建p55PIK慢病毒表达载体Lenti-p55PIK,并观察高表达p55PIK对肿瘤细胞增殖的影响。 方法: 用PCR的方法扩增得到人p55PIK编码区全长片段,通过酶切、连接、转化和质粒提取等步骤得到重组慢病毒表达质粒p55PIK-pCDF,并进行酶切和基因测序鉴定。脂质体转染法在293FT细胞中转染pCDF-p55PIK、pFIV与pVSVG,包装得到可以产生绿色荧光的慢病毒Lenti-p55PIK。将慢病毒Lenti-p55PIK感染肿瘤细胞,用Western blot法检测p55PIK的表达,用流式细胞技术检测增殖细胞相关抗原Ki67的表达,观察p55PIK对细胞增殖的影响。 结果: PCR产物进行琼脂糖凝胶电泳,得到1400bp左右的条带,大小与p55PIK编码基因序列大小一致。重组p55PIK-pCDF质粒进行酶切初步鉴定,证明了p55PIK编码基因序列插入到了pCDF质粒中。经构建好的质粒经酶切初步鉴定后送测序,结果完全符合p55PIK编码基因序列。将pCDF-p55PIK、pFIV与pVSVG转染293FT细胞进行包装,荧光显微镜下可以观察细胞包装效率,裂解细胞得到的病毒Lenti-p55PIK。将病毒感染肿瘤细胞,在荧光显微镜下观察到细胞内显示的绿色荧光,Western blot法检测p55PIK的表达,Lenti-p55PIK组显著高于空病毒组和空白对照组,(P0.01),流式细胞技术检测增殖细胞相关抗原Ki67的表达,结果显示Lenti-p55PIK组显著高于空病毒组和空白对照组,(P0.05)。 结论: 成功构建了人p55PIK慢病毒表达载体Lenti-p55PIK,其能够在肿瘤细胞中高表达人p55PIK蛋白,而且观察到p55PIK促进增殖细胞相关抗原Ki67的表达。
[Abstract]:Aim: to construct p55PIK lentivirus expression vector Lenti-p55PIK and to observe the effect of p55PIK overexpression on the proliferation of tumor cells. Methods: the full-length fragment of human p55PIK coding region was amplified by PCR. The recombinant lentivirus expression plasmid p55PIK-pCDFwas obtained by enzyme digestion, ligation, transformation and plasmid extraction, and identified by enzyme digestion and gene sequencing. PCDF-p55PIKPFIV and pVSVG were transfected into 293FT cells by liposome transfection, and the lentivirus Lenti-p55PIK, which could produce green fluorescence, was packaged. Lenti-p55PIK was infected with Lenti-p55PIK. The expression of p55PIK was detected by Western blot, and the expression of Ki67 was detected by flow cytometry. The effect of p55PIK on cell proliferation was observed. Results: agarose gel electrophoresis showed that the 1400bp band was about the same size as p55PIK coding gene. The recombinant p55PIK-pCDF plasmid was identified by restriction endonuclease digestion. It was proved that p55PIK coding gene sequence was inserted into pCDF plasmid. The constructed plasmids were identified and sequenced by restriction endonuclease digestion, and the results were in good agreement with p55PIK coding gene sequence. PCDF-p55PIKPFIV and pVSVG were transfected into 293FT cells for packaging. The packaging efficiency of the cells and the virus Lenti-p55PIK were observed under fluorescence microscope. The expression of p55PIK in the tumor cells infected with virus was detected by Western blot method under fluorescence microscope. The expression of p55PIK in the Lenti-p55PIK group was significantly higher than that in the empty virus group and the blank control group (P0.01). The expression of Ki67 was detected by flow cytometry. The results showed that Lenti-p55 PIK group was significantly higher than that of empty virus group and blank control group (P0.05). Conclusion: human p55PIK lentivirus expression vector Lenti-p55PIK was successfully constructed, which can overexpress human p55PIK protein in tumor cells, and the expression of p55PIK associated antigen Ki67 was observed.
【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R373

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相关期刊论文 前4条

1 王桂华;罗学来;孙黎;邓豫;李小兰;陶德定;胡俊波;龚建平;;磷脂酰肌醇-3激酶p55γ-N末端24个氨基酸抑制结肠癌细胞增殖的作用[J];癌症;2008年10期

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