蛋白激酶B2慢病毒载体的构建及其沉默效果的验证  

Construction of AKT2 lentivirus vector and validation of its silencing effect

作　　者：戴研平 高晓勤[2] Dai Yanping;Gao Xiaoqin(Department of Pathology and Pathophysiology,Guizhou Medical University,Guiyang 550004;Zunyi Medical and Pharmaceutical College,Zunyi 563006;People's Hospital in Yueyanglou District,Yueyang 414000,China)

机构地区：[1]贵州医科大学基础医学院病理学与病理生理学教研室,贵阳550004 [2]遵义医药高等专科学校基础医学院,遵义563006 [3]岳阳市岳阳楼区人民医院,岳阳414000

出　　处：《解剖学杂志》2021年第2期118-122,共5页Chinese Journal of Anatomy

基　　金：贵州省科学技术基金(黔科合人才团队[2014]4005号)。

摘　　要：目的:利用RNA干扰技术,以蛋白激酶B 2(AKT2)的mRNA为靶点,设计、构建AKT-2的RNA干扰慢病毒载体,并对其沉默效果进行验证。方法:根据目的基因设计3个小干扰RNA(siRNA)靶点,进行引物合成。将单链的引物退火成双链oligo序列,连接入双酶切线性化的RNA干扰载体。测序验证正确的转化子,进行高纯度质粒抽提。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。实验大鼠被随机分为5组,每组10只,空白对照组不注射慢病毒,NC-shRNA阴性感染组、PL-AKT2-shRNA-1感染组、PL-AKT2-shRNA-2感染组、PL-AKT2-shRNA-3感染组。大鼠附睾分别被注射200μL含有NC-shRNA、PL-AKT2-shRNA-1、PLAKT2-shRNA-2、PL-AKT2-shRNA-3的病毒液;免疫印迹检测大鼠附睾AKT2的蛋白沉默效果。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,AKT2的蛋白表达量均较阴性感染组和正常对照组明显降低,其中以PL-AKT2-shRNA-3质粒组干扰效果更为有效。结论:成功设计了AKT-2基因的RNA干扰靶点和制备了AKT-2基因的慢病毒载体,基因沉默效果明显。Objective:To design and construct an AKT2 lentivirus vector with RNA interference targeting AKT2 mRNA,and verify its silencing effect.Methods:Three siRNA targets were designed according to the target gene.Single-stranded primers were annealed into double-stranded oligo sequences and joined into double-enzymatic linearized RNA interference vectors.Sequencing was performed to verify correct inverters and high-purity plasmid extraction was carried out.Three positive cloned plasmids were sequenced and transfected into HEK293-T cells to produce lentivirus plasmids.The experimental rats were randomly divided into 5 groups with 10 rats in each group.The blank control group was not injected with lentivirus.The rats in the NC-shRNA negative infection group,PL-AKT2-shRNA-1 infection group,PL-AKT2-shRNA-2 infection group and PL-AKT2-shRNA-3 infection group were injected in the epididymis with 200μL vectors respectively.Western blotting was used to detect the protein silencing effect of AKT2 in the epididymis of rats.Results:The sequencing results showed that the three lentivirus vectors were packaged successfully.After infection with epididymis,AKT2 protein expression was significantly lower than that in the negative infection group and the normal control group,and the PL-AKT2-shRNA-3 plasmid group was more effective in interfering with AKT2.Conclusion:The RNA interference target of AKT2 gene is successfully designed and the lentivirus vector of AKT2 gene is prepared.The gene silencing effect is obvious.

关 键 词：蛋白激酶B2 慢病毒干扰 基因沉默 附睾 

分 类 号：Q78[生物学—分子生物学]