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1×109 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。%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 × 109 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.
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