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目的构建Pin shRNA慢病毒载体,建立稳定抑制肽基脯氨酰顺反异构酶1(Pin1)表达的A549细胞系。方法根据查阅文献获得Pin1shRNA干扰靶点序列并插入pLenR-GPH载体,构建pLenR-GPH-Pin1shRNA慢病毒载体,随后转入人胚肾HEK293T细胞中,收集其上清进行病毒滴度测定,再行慢病毒的包装,将获得的慢病毒毒液感染A549细胞。通过实时定量PCR(qRT-PCR)及Western blot法检测Pin1在不同组的表达抑制情况。结果经限制性内切酶鉴定及测序分析,成功构建了pLenR-GPH-Pin1shRNA慢病毒载体质粒,包装并得到高滴度的病毒颗粒。通过qRT-PCR和Western blot法检测显示,与对照组相比,pLenR-GPH-Pin1shRNA慢病毒载体感染的A549细胞中Pin1的表达在mRNA和蛋白水平均有所下降。结论慢病毒介导的shRNA能够有效下调A549细胞中Pin 1的表达。
Objective To construct Pin shRNA lentivirus vector and establish A549 cell line stably inhibiting the expression of Pinylproteinase 1 (Pin1). Methods According to the accession literature, Pin1shRNA interference target sequence was obtained and inserted into pLenR-GPH vector to construct lentiviral vector pLenR-GPH-Pin1shRNA, then transferred into human embryonic kidney HEK293T cells. The supernatant was collected for virus titer determination, The virus was packaged and the lentiviral venom obtained was infected A549 cells. The inhibition of Pin1 expression in different groups was detected by real-time quantitative PCR (qRT-PCR) and Western blot. Results The pLenR-GPH-Pin1 shRNA lentiviral vector plasmid was successfully constructed by restriction endonuclease identification and sequencing analysis, and packaged with high titer virus particles. The results of qRT-PCR and Western blot showed that Pin1 expression in A549 cells infected by pLenR-GPH-Pin1 shRNA lentivirus decreased at mRNA and protein levels compared with the control group. Conclusion Lentivirus-mediated shRNA can effectively down-regulate Pin 1 expression in A549 cells.