靶向Rap1b基因shRNA慢病毒载体的构建及其对食管鳞癌细胞Rap1b基因表达的沉默

目的:设计以Rap1b基因为靶点的短发夹状RNA（shRNA），构建重组慢病毒表达载体并转染食管鳞癌细胞，观察其在食管鳞癌细胞中的表达。方法:应用重组DNA 技术，将设计好的4条基因特异性shRNA序列插至慢病毒表达载体pGLV3-GFP中，构建pGLV3-GFP-Rap1b-shRNA1/2/3/4。并应用脂质体法转染293T细胞，进行病毒包装及滴度测定。采用实时荧光定量PCR（RT-PCR）及Western blot分别从mRNA 和蛋白水平检测转染食管鳞癌细胞株Eca109后Rap1b基因的表达情况。结果:测序证实慢病毒载体构建成功，并测定滴度为1×10⁹TU/ml，pGLV3-GFP-Rap1b-shRNA3/4转染后72h和96h均可显著抑制Rap1b基因mRNA及蛋白的表达。结论:成功构建Rap1b基因的shRNA 慢病毒载体，所介导的RNAi能有效抑制食管鳞癌细胞Eca109中Rap1b基因的表达。

Construction of short hairpin RNA lentiviral vector targeting Rap1b gene and its silencing effect on Rap1b gene in esophageal squamous cell carcinoma

To construct lentiviral vector targeting Rap1b gene and evaluate its silencing effect on Rap1b gene in esophageal squamous cell carcinoma. Methods：Four short hairpin RNA（shRNA）fragments targeting Rap1b were designed and cloned into lentiviral vector pGLV3 - GFP to construct pGLV3 - GFP - Rap1b - shRNA1 /2 / 3 / 4. Then the silencing effect on Rap1b gene were confirmed by real - time PCR and Western bolt in transfected gene in esophageal squamous cell carcinoma cell line Eca109. Results：The Rap1b shRNA lentiviral vectors were suc-cessfully constructed confirmed by sequencing and the virus reached a titer of 1 × 10^9 TU/ ml. pGLV3 - GFP - Rap1b- shRNA3 / 4 could significantly inhibit the mRNA and protein expression of Rap1b among four shRNA fragments. Conclusion：shRNA expressing lentiviral recombinants targeting the Rap1b gene were successfully constructed,which had silencing effect on Rap1b gene in esophageal squamous cell carcinoma cell line Eca109.