靶向YWHAE基因shRNA慢病毒表达质粒的构建及功能验证

发布时间：2018-04-25 21:15

  本文选题：慢病毒属 + RNA干扰　； 参考：《福建医科大学学报》2017年03期

【摘要】：目的构建靶向YWHAE基因的shRNA慢病毒表达质粒,并验证其对AGS胃癌细胞增殖的影响。方法设计并合成5对针对人YWHAE基因的shRNA序列,分别克隆入pLentiLox3.7(pLL3.7)慢病毒表达质粒,接着将重组的慢病毒表达质粒和包装质粒PHR、包膜质粒VSVG一起采用磷酸钙法转染293T细胞包装慢病毒,收集制备的慢病毒感染AGS细胞,用抗生素Puromycine进行筛选。Western-blot检测感染细胞YWHAE蛋白的表达。选取干扰效率最高的慢病毒表达质粒,针对性设计siRNA的点突变引物,重叠延伸PCR法扩增获得点突变的YWHAE基因,克隆入pcDNA3.1/myc-His(-)A载体,构建YWHAE点突变的表达质粒,转染YWHAE沉默效果最好的AGS细胞株,Western-blot检测转染细胞YWHAE蛋白的回复表达。采用MTS法检测YWHAEshRNA慢病毒对AGS细胞增殖的影响。结果5对针对人YWHAE基因的shRNA序列构建的慢病毒中,pLL3.7-siYWHAE-5包装成的慢病毒抑制AGS细胞的YWHAE蛋白表达的效果最明显,仅为对照组相对表达量的(0.269±0.083)倍;pLL3.7-siYWHAE-5包装的慢病毒,其干扰效应可经由YWHAE点突变表达质粒回复。与对照组比较,YWHAE-shRNA组AGS细胞增殖能力明显下降。结论成功构建了靶向YWHAE基因的shRNA慢病毒表达质粒,获得YWHAE基因表达显著下调的AGS细胞株;探明YWHAE表达的下调可有效抑制AGS细胞的增殖,提示YWHAE在胃癌中的致癌潜能。
[Abstract]:Objective to construct the shRNA lentivirus expression plasmid targeting YWHAE gene and verify its effect on the proliferation of AGS gastric cancer cells. Methods five pairs of shRNA sequences targeting human YWHAE gene were designed and synthesized and cloned into lentivirus expression plasmids pLL3.7pL3.7pL3.7pL3.7pL3.7. the recombinant lentiviral expression plasmids and packaging plasmids were then transfected into 293T cells by calcium phosphate method with encapsulated plasmid VSVG. The lentivirus was collected and infected with AGS cells. The expression of YWHAE protein in infected cells was detected by Puromycine screening. The point mutation primers of siRNA were designed by using the lentivirus expression plasmid with the highest interference efficiency. The point mutant YWHAE gene was amplified by overlapping extension PCR and cloned into the pcDNA3.1/myc-His(-)A vector to construct the point mutation expression plasmid of YWHAE. Western-blot was used to detect the expression of YWHAE protein in transfected AGS cells. The effect of YWHAEshRNA lentivirus on the proliferation of AGS cells was detected by MTS assay. Results 5 lentiviruses packaged with lentivirus pL3.7-siYWHAE-5 targeting the shRNA sequence of human YWHAE gene had the most obvious effect on inhibiting the expression of YWHAE protein in AGS cells, only 0.269 卤0.083 times of the relative expression of pLL3.7-siYWHAE-5 in the control group. Its interference effect can be recovered by YWHAE point mutation expression plasmid. Compared with the control group, the proliferation ability of AGS cells in YWHAE-shRNA group was significantly decreased. Conclusion the expression plasmid of shRNA lentivirus targeting YWHAE gene was successfully constructed and the AGS cell line with significantly down-regulated expression of YWHAE gene was obtained. It was found that the down-regulation of YWHAE expression could effectively inhibit the proliferation of AGS cells and suggest the carcinogenic potential of YWHAE in gastric cancer.
【作者单位】： 福建医科大学消化道恶性肿瘤教育部重点实验室福建省肿瘤微生物重点实验室;
【基金】：国家自然科学基金(81271784) 福建省教育厅项目(JA12150) 福建医科大学重大科研项目基金(09ZD018);福建医科大学苗圃科研基金(2010MP029)
【分类号】：R735.2
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本文编号：1802995