shRNA-PAX6慢病毒载体构建及其对胶质瘤U251细胞增殖的影响

发布时间：2018-07-07 16:42

本文选题：PAX基因 + 胶质瘤细胞　；参考：《南方医科大学学报》2017年12期

【摘要】：目的构建shRNA-PAX6慢病毒载体并观察其对胶质瘤U251细胞增殖的影响。方法根据文献报道的PAX6靶点序列,设计两条PAX6基因的si RNA靶点干扰序列,引物退火形成带粘性末端的双链,与经BamH I、EcoR I双酶切后线性化的慢病毒载体进行连接、转化,构建shRNA-PAX6慢病毒重组载体,再经菌落PCR及测序鉴定重组载体;在293T细胞中包装病毒,将包装后的shRNA-PAX6慢病毒重组载体感染U251细胞。Real-time PCR检测PAX6 mRNA的表达水平,Western blot检测PAX6蛋白的表达,MTT法检测U251细胞增殖。结果慢病毒载体p LKD-CMV-GNR-U6-shRNA经双酶切后可见线性化的8208 bp大片段,菌落PCR及测序鉴定证实shRNA-PAX6慢病毒重组载体构建成功,构建的shRNA-PAX6慢病毒载体在293T细胞完成包装,测得慢病毒滴度为6.73×10~8TU/m L;Real-time PCR结果显示,沉默PAX6的表达可见U251细胞PAX6 mRNA表达水平明显低于正常对照组及慢病毒空载体组(P0.05);Western blot结果显示,PAX6蛋白表达亦明显低于正常对照组及慢病毒空载体组(P0.05);MTT结果显示,与正常对照组及慢病毒空载体组细胞比较,感染shRNA-PAX6慢病毒重组载体组的细胞,细胞增殖能力明显增强(P0.05)。结论成功构建人PAX6基因的shRNA-PAX6慢病毒重组载体,沉默PAX6基因的表达,U251细胞的增殖能力增强。
[Abstract]:Objective to construct shRNA-PAX6 lentivirus vector and to observe the effect of shRNA-PAX6 lentivirus vector on the proliferation of glioma U251 cells. Methods according to the PAX6 target sequence reported in the literature, two si RNA target interference sequences of PAX6 gene were designed. The primers were annealed to form double strands with sticky ends, and ligated with lentivirus vector linearized by BamH Irito Ecor I double enzyme. The recombinant vector of lentivirus shRNA-PAX6 was constructed, and the recombinant vector was identified by colony PCR and sequencing. The expression level of PAX6 mRNA was detected by real-time PCR in U251 cells infected with recombinant shRNA-PAX6 lentivirus vector. The expression of PAX6 protein was detected by Western blot and the proliferation of U251 cells was detected by MTT assay. Results A linearized 8208 BP fragment of lentivirus vector pLKD-CMV-GNR-U6-shRNA was found after double enzyme digestion. Colony PCR and sequencing confirmed that the recombinant shRNA-PAX6 lentivirus vector was successfully constructed, and the constructed shRNA-PAX6 lentivirus vector was packaged in 293T cells. The titer of lentivirus was 6.73 脳 10 ~ (-8) TU / m ~ (real-time) PCR. The expression of PAX6 mRNA in U251 cells was significantly lower than that in normal control group and lentivirus empty vector group (P0.05). The results of Western blot showed that the expression of PAX6 protein in U251 cells was significantly lower than that in normal control group and lentivirus empty vector group (P0.05). Compared with the normal control group and the lentivirus empty vector group, the cells infected with shRNA-PAX6 lentivirus recombinant vector group, the cell proliferation ability was significantly increased (P0.05). Conclusion the recombinant vector of human PAX6 gene shRNA-PAX6 lentivirus was successfully constructed and the proliferation ability of U251 cells was enhanced by silencing the expression of PAX6 gene.
【作者单位】：中南大学湘雅医学院生理学系;
【基金】：国家自然科学基金(81472160) 湖南省自然科学基金(2015JJ2157)~~
【分类号】：R739.41

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