siRNA前体慢病毒干扰A549细胞株蛋白激酶2a表达的载体构建及鉴定

目的构建针对蛋白激酶2a（caseinkinase2a，CK2a）基因的siRNA前体（shortharpinRNA，shRNA）表达载体，鉴定CK2a基因干扰效率。方法体外设计并合成针对CK2a基因的慢病毒shRNA干扰载体，通过PCR及测序证实载体构建成功。将人肺腺癌细胞株A549分为3组，病毒干扰组（CK组）将慢病毒颗粒以最适滴度感染细胞株A549，病毒空载组（NC组）将慢病毒颗粒空载体感染细胞株A549，阴性对照组（CON组）为未经任何处理的细胞株A549；检测各组CK2a的干扰效率。结果构建的慢病毒载体序列通过PCR、测序等鉴定证实与设计序列相同；实时定量PCR检测显示CK组A549细胞CK2amRNA表达率较NC组及CON组下降约80％；Westernblotting显示CK组A549细胞表达CK2a蛋白量较NC组及CON组明显减少。结论构建的shRNA表达载体可在mRNA和蛋白水平上有效抑制A549细胞株CK2a基因的表达。

Construction and identification of protein kinase CK2a gene expression in A549 cell line by using lentivirus-mediated short harpin RNA

Objective To construct the short harpin RNA （shRNA） expression vector targeting protein kinase CK2a gene and to indentify its silencing effect on human lung cancer cell line A549. Methods Three groups of target segments were synthesized and cloned into lentivirus-mediated shRNA respectively and the vectors were identified by enzyme digestion analysis and DNA sequencing. Then the expression vectors were transfected into A549 cell line （CK group）, the empty vectors were transfected into A549 cell line （NC group）, and those received no treatment of A549 cell line were negative control group （CON group）. The expressions of CK2a at mRNA and protein levels were detected. Results Enzyme digestion analysis and DNA sequencing showed that the target segments were cloned into pSilence 4. 1 lentiviral vector respectively. Real-time PCR detected that the expression rate of CK2a gene of A549 cells in CK group dropped by about 80~ compared with NC group and CON group. Western blotting also showed that the expression of CK2a protein decreased in CK group more obviously than that in NC group and CON group. Conclusion The shRNA expression vector can effectively inhibit the expression of CK2a gene at mRNA and protein levels in A549 cell line.