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目的构建针对蛋白激酶2a(casein kinase 2a,CK2a)基因的siRNA前体(short harpin RNA,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%;Western blotting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。
Objective To construct a short hairpin RNA (shRNA) expression vector targeting protein kinase 2a (CK2a) gene and identify the interference efficiency of CK2a gene. Methods Lentiviral shRNA targeting CK2a gene was designed and synthesized in vitro. The vector was successfully constructed by PCR and sequencing. The human lung adenocarcinoma cell line A549 was divided into three groups. The virus-interfering group (CK group) infected lentivirus particles with the optimal titer to A549 cell line, Strain A549, negative control group (CON group) without any treatment cell line A549; CK2a detection efficiency of each group. Results The constructed lentiviral vector sequence was verified by PCR, sequencing and the same sequence as the designed sequence. Real-time quantitative PCR showed that CK2amRNA expression rate of A549 cells in CK group decreased about 80% compared with NC and CON groups; Western blotting showed that A549 cells were expressed in CK group CK2a protein content than the NC group and CON group was significantly reduced. Conclusion The constructed shRNA expression vector can effectively inhibit the expression of CK2a gene in A549 cell line at mRNA and protein levels.