DGKζ基因RNA干扰慢病毒载体制备及感染结肠癌RKO细胞的干扰效应

摘要

目的 应用RNA干扰技术构建DGKζ基因敲减慢病毒载体,感染结肠癌RKO细胞,观察其抑制DGKζ表达的效率.方法 运用Real-time PCR及Western blot检测8株结肠癌细胞中DGKζ基因的表达.针对DGKζ基因序列,设计RNA干扰靶点序列,合成含干扰序列的双链DNA oligo,慢病毒载体酶切,转染备好的细菌感受态细胞,对长出的克隆先进行PCR鉴定,再进行测序比对后,鉴定阳性的克隆即为构建成功的DGKζ基因RNA干扰慢病毒载体.慢病毒感染RKO细胞后,运用Real time PCR和Western blot法检测DGKζ基因的敲减效率.结果 8株结肠癌细胞株中,DGKζ在RKO细胞中的表达丰度最高,满足敲减要求;测序表明重组质粒构建成功;Real-time PCR实验结果显示,在RKO细胞中,相对于感染慢病毒空载体细胞,DGKζ基因的敲减效率均达到70％以上(P＜0.05).Western blot结果显示,RKO细胞和感染慢病毒空载体细胞(Negative control)在124kDa处出现免疫印迹,而干扰敲减DGKζ基因的细胞(shRNA-DGKζ)则不表达内源性DGKζ蛋白,表明DGKζ基因慢病毒感染有很高的敲减效率.结论 成功构建DGKζ基因敲减慢病毒载体,进一步证实了感染结肠癌RKO细胞的特异性干扰效应.%Objective To construct the DGK ζ knock-down lentivirus vector by using RNA interference technique,and to infect the RKO colon cancer cells.Methods The expression pattern of DGK ζ mRNA in eight colon cancer cell lines was detected by using real-time PCR.Based on the coding sequence of DGK ζ,the RNA interference target sequence was designed and the double-stranded DNA oligo and lentivirus vector enzyme digestion was synthetized.An efficient bacterial transformation system was used for clone PCR identification.After sequence was tested and contrasted,the positive clone was taken as DGK ζ knock-down lentivirus vector.The efficiency of DGK ζ were validated by real time PCR and Western blot analysis.Results The highest expression abundance of DGK ζ mRNA was showed in RKO cells.Sequence analysis revealed that the recombinant plasmid was constructed successfully.Real time PCR analysis indicated that the DGK ζ knock down efficiency was achieved 70％ compared to the negative control (P＜0.05).Western blot analysis indicated that the DGK ζ knock-down lentivirus vector infection possess high efficiency.Conclusion Construct the DGK ζ knock-down lentivirus vector successfully and further verifies the specific interference effect for infection of human RKO colon cancer cells.