CASK基因过表达慢病毒载体的构建及鉴定

Construction and identification of lentiviral vector over-expressing CASK gene

目的:构建人钙/钙调蛋白依赖性丝氨酸蛋白激酶(CASK)基因过表达慢病毒载体并进行鉴定。方法:采用聚合酶链反应(PCR)扩增CASK基因片段,通过连接酶将CASK基因片段连接至线性化的GV358载体上,运用PCR方法鉴定阳性克隆的CASK-GV358载体;将重组质粒和包装质粒共转染至293T细胞,并用酶联免疫吸附试验(ELISA)法进行滴度检测。将成功包装的CASK过表达慢病毒LV-CASK(OE组)和阴性对照病毒CON238(NC组)分别感染人非小细胞肺癌(NSCLC)H1299细胞,同时设空白对照组(MOCK组),用荧光定量PCR(FQ-PCR)和western blotting法检测CASK表达水平。结果:通过PCR技术成功地扩增CASK基因片段并连接到GV358病毒载体上,PCR及DNA测序鉴定结果证明CASK-GV358质粒构建正确;重组慢病毒转染293T细胞后可观察到绿色荧光及蛋白表达,包装过表达慢病毒并测定其浓缩滴度为2×108TU/mL。FQ-PCR和western blotting检测结果显示,与NC组相比,OE组CASK mRNA和蛋白的表达均显著上调(P<0.05)。结论:成功构建CASK基因过表达慢病毒载体,可在H1299细胞中稳定表达,为CASK基因的后期研究提供了实验基础。

Objective： To construct a lentiviral vector over-expressing calcium/calmodulin-dependent serine protein kinase （CASK）. Methods： The whole length CASK gene fragment was amplified by polymerase chain reaction （PCR） and inserted into linearized GV358 lentiviral vectors using ligase. Positive clones of CASK-GV358 vectors were identified by PCR. The lentiviral vectors containing CASK gene were co-transfected into 293T cells with packaging plasmids. The virus titer was determined by ELISA. The recombinant lentivirus over-expressing CASK （LV-CASK） and negative control recombinant lentivirus CON238 were used to infect human NSCLC H1299 ceils, respectively. Fluorescence quantitative PCR （FQ-PCR） and western blotting were used to detect the expression of CASK. Results： CASK gene was amplified and successfully inserted into the GV358 lentivirus vector. The sequences of the recombinant plasmid were confirmed by PCR and DNA sequencing. The expression of GFP could be detected in 293T cells infected with recombinant lentiviruses. The lentiviruses over-expressing CASK were harvested and concentrated to 2× 10^8 TU/mL. According to the results of FQ-PCR and western blotting, the mRNA and protein expression level of CASK significantly upregulated in H1299 cell infected with LV-CASK, compared to cells infected with CON238 （ P 〈0.05）. Conclusion： The lentivriral vector over-expressing CASK gene was constructed successfully and stably expressed CASK in H1299 cells.