Beclin1基因慢病毒表达载体的构建和鉴定

背景：Beclin 1基因是哺乳动物的自噬调控基因。目的：实验拟构建Beclin 1基因慢病毒过表达载体。方法：聚合酶链反应扩增目的基因Beclin 1后插入慢病毒表达载体pLenex中,构建重组载体pLenex-Beclin 1。使用聚合酶链反应、双酶切和DNA的测序方法对其进行鉴定,并与辅助包装质粒共感染293T细胞。慢病毒颗粒转染非小细胞肺癌A549细胞后,用蛋白质印迹法检测Beclin 1基因的过表达效率。结果与结论：聚合酶链反应鉴定结果显示扩增的阳性片段已插入pLenex载体,聚合酶链反应、双酶切和DNA测序结果表明,重组慢病毒载体pLenex-Beclin 1的插入序列完全正确,重组慢病毒载体感染A549细胞后,细胞内Beclin 1蛋白高效表达。结果证实,实验成功构建了Beclin 1基因慢病毒过表达载体。

Construction and identification of lentiviral vector for Beclin1 gene

BACKGROUND：Beclinl is an essential autophagy gene.OBJECTIVE：To construct lentiviral vector pLenex-Beclin1 gene.METHODS：Beclin1 gene amplification was used by real-time polymerase chain reaction.Gene amplification products were inserted in the lentiviral vector pLenex,and constructed lentiviral vector pLenex-Beclin1.Polymerase chain reaction analysis,double digests and DNA sequencing were used to confirm the constructed vectors whether or not success recombinant vector plasmid and to coinfect 293T cells,and then was transfected into non small cell lung cancer A549 cells.Western blot was then used to investigate the interfering efficiency of Beclin1 gene.RESULTS AND CONCLUSION：Polymerase chain reaction tests showed amplified positive fragments were inserted in pLenex vectors.Results of polymerase chain reaction analysis,double digestion and DNA sequencing showed that recombinant lentivirus plasmids pLenex-Beclin1 were constructed successfully.In transfected A549 cells,Beclin1 protein was over-expressed.Results verified that lentiviral vectors of Beclin1 gene over-expression were successfully constructed.