RNA干扰USE1基因慢病毒载体的构建及鉴定

doi:10.13560/j.cnki.biotech.bull.1985.2018-0659

摘要/Abstract

摘要： 旨在构建泛素结合酶USE1基因的RNA干扰慢病毒载体,并在细胞中检测该基因的抑制表达水平,以期寻找Uba6-USE1特异性泛素化通路对应的下游底物并研究其功能。选取并合成靶向USE1基因的特异性shRNA序列,将其克隆至pLL3.7慢病毒抑制表达载体上,构建抑制USE1基因表达的重组慢病毒质粒并鉴定。将鉴定成功的重组质粒与psPAX2、VSVG慢病毒包装载体共转染至HEK-293细胞,收集病毒上清,测定病毒滴度并确定最佳感染稀释倍数,通过qPCR和Western Blot方法检测病毒感染HEK-293细胞后对USE1基因的抑制程度,获得能有效抑制USE1基因的慢病毒上清。结果显示,成功构建3种靶向USE1基因的RNA干扰慢病毒载体,获得滴度符合要求的3种慢病毒包装上清液,感染HEK-293细胞后发现,第2、3号shRNA序列均有明显抑制表达USE1基因效果,基因表达抑制率约50%（^*P<0.05）。利用RNA干扰技术成功构建了特异性抑制泛素结合酶USE1基因表达的慢病毒载体,并经mRNA和蛋白水平检验得到2种能够显著抑制USE1表达的慢病毒上清及其shRNA序列。

关键词: 泛素结合酶USE1, RNA干扰, 慢病毒载体, 泛素化

Abstract: This work aims to construct a lentiviral vector for RNA interference of ubiquitin-conjugating enzyme USE1 gene and to detect the inhibition level of this gene in cells for identifying the downstream substrates corresponding to the specific ubiquitination pathway of Uba6-USE1. The specific shRNA sequence targeting USE1 gene were selected,synthesized,and cloned into pLL3.7 lentiviral vector,and the recombinant lentiviral plasmids inhibiting the expression of USE1 gene were constructed and identified. The identified recombinant plasmids were co-transfected into HEK-293 cell with psPAX2 and VSVG lentiviral packaging vectors. Then viral supernatants were collected and the titers as well as the optimal dilution factors of the virus were determined. The inhibition of the USE1 gene in viral infected HEK-293 cells was detected by qPCR and Western Blot. Results showed that 3 RNA-interfered lentiviral vectors targeting USE1 gene were constructed successfully,the supernatants of 3 lentiviral packaging vectors with satisfied titers were collected. Number 2 and 3 shRNA sequences presented obvious inhibitions to the expression of USE1 gene with an inhibition rate at 50%（^*P<0.05）. In sum,lentiviral vectors specifically inhibiting the expression of ubiquitin-conjugating enzyme USE1 gene are successfully constructed by RNA interference,and supernatants and shRNA sequences significantly inhibiting the expression of USE1 are obtained after mRNA and protein verification.

Key words: ubiquitin-conjugating enzyme USE1, RNA interference, lentiviral vector, ubiquitination

王佳悦, 刘香男, 彭康莉, 赵博. RNA干扰USE1基因慢病毒载体的构建及鉴定[J]. 生物技术通报, 2019, 35(3): 117-122.

WANG Jia-yue, LIU Xiang-nan, PENG Kang-li, ZHAO Bo. Construction and Identification of Lentiviral Vector for RNA Interference of USE1 Gene[J]. Biotechnology Bulletin, 2019, 35(3): 117-122.

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