附睾血管内皮生长因子基因慢病毒干扰载体的构建  

Construction of lentiviral interference vector of epididymal vascular endothelial growth factor gene

作　　者：戴研平 王平[2] 高晓勤[2] Dai Yanping;Wang Ping;Gao Xiaoqin(School of Medicine,Yueyang Vocational and Technical College,Yueyang 414000;Department of Zunyi Medical and Pharmaceulical College,Zunyi 563006,China)

机构地区：[1]岳阳职业技术学院医学院,岳阳414000 [2]遵义医药高等专科学校基础医学院,遵义563006

出　　处：《解剖学杂志》2022年第5期445-448,共4页Chinese Journal of Anatomy

摘　　要：目的:以血管内皮生长因子(VEGF)的mRNA为靶点,设计、构建VEGF的RNA干扰慢病毒载体,并对其在大鼠附睾的沉默效果进行验证。方法:构建3种附睾VEGF-shRNA慢病毒表达载体VEGF-shRNA-1、VEGF-shRNA-2、VEGF-shRNA-3,提取3种阳性克隆质粒测序,转染HEK293-T细胞后产生慢病毒质粒。大鼠随机分为空白对照组、NC-shRNA阴性感染组、VEGF-shRNA-1感染组、VEGF-shRNA-2感染组、VEGF-shRNA-3感染组,将病毒液分别注射到各组大鼠双侧附睾;实时荧光定量PCR及蛋白免疫印迹检测各组大鼠附睾VEGF的mRNA及蛋白沉默效果。结果:测序结果证明3种慢病毒载体被包装成功。感染大鼠附睾后,VEGF感染组mRNA及蛋白表达量均较阴性感染组和空白对照组明显降低,其中以VEGF-shRNA-3感染组干扰效果更为有效。结论:成功设计了VEGF基因的RNA干扰靶点和制备了VEGF基因的慢病毒载体,VEGF-shRNA-3能有效抑制附睾VEGF的表达。Objective:To design and construct vascular endothelial growth factor(VEGF)lentivirus vectors with RNA interference(RNAi) targeting VEGF mRNA,and verify its silencing effect in rat epididymis.Methods:RNA interference(RNAi) was used to target VEGF mRNA,VEGF-shRNA-1,VEGF-shRNA-2 and VEGF-shRNA-3 expression vectors containing epididymal VEGF-shRNA lentivirus were constructed,and three positive cloned plasmids were extracted and sequenced.Following transfection of the plasmids into human embryonic kidney(HEK) 293-T cells,rats were randomly divided into 5 groups(control group,NC-shRNA infection group,VEGF-shRNA-1 infection group,VEGF-shRNA-2 infection group,VEGF-shRNA-3 infection group),and 200 μL of recombinant lentiviruses and negative control lentiviruses were injected into the bilateral epididymis of each group of rats.Quantitative real-time PCR(RT-qPCR)and Western blotting were performed to detect the mRNA and protein silencing effect of VEGF in the epididymis of rats.Results:The sequencing results showed that three lentivirus vectors were packaged successfully.After infection with epididymis,VEGF mRNA and protein expression levels in VEGF-shRNA infection groups were significantly lower than that in the negative infection group and the normal control group,and the VEGF-shRNA-3 plasmid group was more effective in interfering with VEGF.Conclusion:The RNA interference target of VEGF gene was successfully designed and the lentivirus vector of VEGF gene was prepared.VEGF-shRNA-3 has the best interference effect and could effectively inhibit the expression of VEGF in the epididymis.

关 键 词：慢病毒干扰 血管内皮生长因子 基因沉默 附睾 大鼠 

分 类 号：R-33[医药卫生] R698.2