中文摘要：

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1×10^9 TU/ 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。

英文摘要：

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.