RNA干扰USE1基因慢病毒载体的构建及鉴定
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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。
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 m RNA and protein verification.
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 m RNA and protein verification.
引文
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[2]PickartCM,EddinsMJ.Ubiquitin:structures,functions,mechanisms[J]. Biochimica Biophysica Acta. Molecular Cell Research, 2004, 1695(3):55-72.
[3]Pickart CM. Back to the future with ubiquitin[J]. Cell, 2004, 116(2), 181-190.
[4]Jin J, Li X, Gygi SP, et al. Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging[J]. Nature, 2007, 447(7148):1135-1138.
[5]Chiu YH, Sun Q, Chen ZJ. E1-L2 activates both ubiquitin and FAT10[J]. Mol Cell, 2007, 27:1014-1023.
[6]Pelzer C, Kassner I, Matentzoglu K., et al. UBE1L2, a novel E1enzyme specific for ubiquitin[J]. Journal of Biological Chemistry,2007, 282(32):23010-23014.
[7]Metzger MB, Hristova VA, Weissman AM. HECT and RING finger families of E3 ubiquitin ligases at a glance[J]. Journal of Cell Science, 2012, 125(Pt 3):531.
[8]Zhou MJ, Chen FZ, Chen HC. Ubiquitination involved enzymes and cancer[J]. Medical Oncology, 2014, 31(8):93.
[9]Zhao B, Bhuripanyo K, Zhang KY, et al. Orthogonal ubiquitin transfer through engineered E1-E2 cascades for protein ubiquitination[J]. Chemistry&Biology, 2012, 19(10):1265-1277.
[10]Liu X, Zhao B, et al. Orthogonal ubiquitin transfer identifies ubiquitination substrates under differential control by the two ubiquitin activating enzymes[J]. Nat Commun, 2017, 8:14286.
[11]Kim SJ, Lee TH, Sang HN, et al. Association of Uba6-Specific-E2(USE1)With Lung Tumorigenesis[J]. Journal of the National Cancer Institute, 2016, 109(3):1-11.
[12]Hershko A, Ciechanover A. The ubiquitin system[J]. Annual Review of Biochemistry, 1998, 67:425-479.
[13]王佳悦,赵博.泛素化在肿瘤治疗方面的应用及展望[J].生命的化学, 2017, 37(6):879-887.
[14]何伟珍,徐晓东.泛素-蛋白酶体途径与疾病[J].海南医学,2007, 18(3):141-142.
[15]唐珍,罗蓉.泛素-蛋白酶体通路与疾病发生的研究进展[J].兰州大学学报:医学版, 2014, 40(3):71-75.
[16]Dou QP, Zonder JA. Overview of proteasome inhibitor-based anti-cancer therapies:perspective on bortezomib and second generation proteasome inhibitors versus future generation inhibitors of ubiquitin-proteasome system[J]. Current Cancer Drug Targets, 2014, 14(6):517-536.
[17]Sumimoto H, Kawakami Y. Lentiviral vector-mediated RNAi and its use for cancer research[J]. Future Oncol, 2007, 3(6):655-664.
[18]Katayama K, Koichiro W, Miyoshi H, et al. RNA interfering approach for clarifying the PPAR pathway using lentiviral vectors expressing short hairpin RNA[J]. FEBS Letters, 2004, 560(3):178-182.
[19]Kafri T, Praag HV, Ouyang L, et al. A Packaging cell line for lentivirus vectors[J]. Journal of Virology, 1999, 73(1):576.
[20]Naldini L, Bl?mer U, Gallay P, et al. In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector[J].Science, 1996, 272(5259):263-267.
[21]张景海,杨保胜,颜真,等.药学分子生物学[M].第4版.北京:人民卫生出版社, 2013.