β-arrestin 2 shRNA慢病毒载体的构建及鉴定

Construction and verification of a lentivector for beta arrestin 2 short hairpin RNA

目的构建β-arrestin 2的shRNA慢病毒载体,获得稳定低表达β-arrestin 2的人非小细胞肺癌A549细胞并验证敲低效果。方法查询4对可信的β-arrestin 2敲低序列,将载体质粒pSicoR-GFP线性化,构建目的质粒,转化感受态细胞,对长出的克隆应用菌落PCR鉴定,再对阳性结果进行测序和比对分析。重组病毒质粒与另外三种辅助包装原件载体质粒通过Lipofectamine 2000共转染293T细胞,培养48 h后,收集细胞培养上清液,导入人非小细胞肺癌细胞A549中,用qRT-PCR检测转染效率。结果成功构建β-arrestin 2的shRNA慢病毒载体质粒,测序比对结果一致。qRT-PCR检测发现一组β-arrestin 2的mRNA表达只有对照组的56%。结论成功构建β-arrestin 2的shRNA慢病毒载体,并验证其在人非小细胞肺癌细胞A549中敲低效果显著。

Objective To construct and verify the p-arrestin 2 shRNA lentivector. Methods Four credible p-arrestin 2 knock-down sequences were searched. The linearized lentiviral vector pSicoR-GFP and shRNAs were constructed into aim plasmid pSicoR-shRNA under the effect of In-Fusion convertase. The aim plasmids were transfected into competent cells. The grown colonies were identified by colony PCR, and then the positive colonies were sequenced. Aligned recombinant lentivector plasmids and the other three helping plasmids were cotransfected into 293T cells by Lipofectamine 2000, and cell culture supernatant after 48 h of culture was collected to treat human non- small cell lung cancer cells A549, then the transfection efficiency was detected by qRT-PCR. Results p-arrestin shRNA lentivirus vector plasmid was constructed successfully, and the sequencing comparison results were consistent. qRT-PCR showed that one group of the β-arrestin 2 expression was only 56% of the control group. Condusion The p-arrestin 2 shRNA lentivector has been successfully constructedand its knock-down efficiency was verified in human non-small cell lung cancer cells A549.