靶向3D基因shRNA慢病毒载体的构建及功能鉴定
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摘要
本研究旨在筛选抑制B组柯萨奇病毒(coxsackievirus B,CVB)3D聚合酶表达的短发卡RNA(short hairpin RNA,shRNA)并构建其慢病毒表达载体,以抑制CVB的3D聚合酶表达。设计并合成3对针对3D基因的RNA干扰序列及相应的对照组序列,通过定量反转录-聚合酶链反应(quantitative reverse transcription-polymerase chain reaction,RT-qPCR)和蛋白免疫印迹法筛选出干扰效率最高的一对,合成shRNA序列。退火后,应用基因重组技术构建pLVTHM-3DshRNA重组质粒,经酶切鉴定及DNA测序后,将重组正确的pLVTHM-3DshRNA与psPAX2、pMD2.G共转染293T细胞,包装慢病毒并鉴定其对CVB3 3D基因表达的干扰效果。测序结果显示,成功构建了pLVTHM-3DshRNA重组慢病毒载体,并收获了包装后的慢病毒,病毒滴度为5×10~(7 )TU。感染HeLa细胞和BALB/c小鼠后,3D聚合酶表达量下降。本研究成功构建了慢病毒Lenti-sh3D,其能有效下调CVB 3D聚合酶的表达,为进一步抗CVB致病机制的研究奠定了基础。
The present paper aimed to inhibit the expression of 3 D polymerase of coxsackievirus B by short hairpin RNAs(shRNAs) with lentiviral vector. Three pairs of RNA interference sequences targeting 3 D gene and corresponding control sequences were designed and synthesized. The pair with the highest interference efficiency was screened by quantitative reverse transcription-polymerase chain reaction(RT-qPCR) and Western blotting. The recombinant plasmid pLVTHM-3 DshRNA was constructed and co-transfected with psPAX2 and pMD2.G in 293 T cells. The packaged lentivirus(titer 5×10~7 TU) was subjected to functional evaluations in HeLa cells and mice. The inhibition of 3 D polymerase expression was observed.
The present paper aimed to inhibit the expression of 3 D polymerase of coxsackievirus B by short hairpin RNAs(shRNAs) with lentiviral vector. Three pairs of RNA interference sequences targeting 3 D gene and corresponding control sequences were designed and synthesized. The pair with the highest interference efficiency was screened by quantitative reverse transcription-polymerase chain reaction(RT-qPCR) and Western blotting. The recombinant plasmid pLVTHM-3 DshRNA was constructed and co-transfected with psPAX2 and pMD2.G in 293 T cells. The packaged lentivirus(titer 5×10~7 TU) was subjected to functional evaluations in HeLa cells and mice. The inhibition of 3 D polymerase expression was observed.
引文
[1] Zhai X, Wu S, Lin X, Wang T, Zhong X, Chen Y, Xu W, Tong L, Wang Y, Zhao W, Zhong Z. Stress granule formation is one of the early antiviral mechanisms for host cells against coxsackievirus B infection [J]. Virol Sin, 2018, 33: 314-322.
[2] Kim DS, Nam JH. Characterization of attenuated coxsackievirus B3 strains and prospects of their application as live-attenuated vaccines [J]. Expert Opin Biol Ther, 2010, 10(2): 179-190.
[3] 汤晶晶, 张杰, 李凯, 樊帆, 李琼芬, 丁峥嵘.云南省2011—2014年急性弛缓性麻痹病例非脊髓灰质炎肠道病毒分子流行病学特征 [J]. 中国疫苗和免疫, 2017, 23(6): 608-612,646.
[4] Andréoletti L, Hober D, Becquart P, Belaich S, Copin MC, Lambert V, Wattré P. Experimental CVB3-induced chronic myocarditis in two murine strains: evidence of interrelationships between virus replication and myocardial damage in persistent cardiac infection [J]. J Med Virol, 1997, 52(2): 206-214.
[5] Lindberg AM, St?lhandske PO, Pettersson U. Genome of coxsackievirus B3 [J]. Virology, 1987, 156(1): 50-63.
[6] Kempf BJ, Barton DJ. Picornavirus RNA polyadenylation by 3Dpol, the viral RNA-dependent RNA polymerase [J]. Virus Res, 2015, 206: 3-11.
[7] Xu HT, Hassounah SA, Colby-Germinario SP, Oliveira M, Fogarty C, Quan Y, Han Y, Golubkov O, Ibanescu I, Brenner B, Stranix BR, Wainberg MA. Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors [J]. J Antimicrob Chemother, 2017, 72(3): 727-734.
[8] 张秀杰, 韩晓峰, 欧阳红生. HCV治疗的新方法:RNA依赖的RNA聚合酶抑制剂[J]. 生命的化学, 2007, 27(3): 251-253.
[9] Mondal M, Klimov P, Flynt AS. Rewired RNAi-mediated genome surveillance in house dust mites [J]. PLoS Genet, 2018, 14(1): e1007183.
[10] 王永佳, 陈永胜, 李国瑞. RNAi在植物中的作用机理及其应用研究进展[J]. 内蒙古民族大学学报(自然汉文版), 2010, 25(1): 54-58.
[11] MacKeigan JP, Murphy LO, Blenis J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance [J]. Nat Cell Biol, 2005, 7(6): 591-600.
[12] Frank SB, Schulz VV, Miranti CK. A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector [J]. BMC Biotechnol, 2017, 17(1): 24. doi: 10.1186/s12896-017-0341-x.
[13] Poling BC, Tsai K, Kang D, Ren L, Kennedy EM, Cullen BR. A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs [J]. RNA Biol, 2017, 14(11): 1570-1579.
[14] Rao DD, Vorhies JS, Senzer N, Nemunaitis J. siRNA vs. shRNA: similarities and differences [J]. Adv Drug Deliv Rev, 2009, 61(9): 746-759.
[15] Li M, Rossi JJ. Lentiviral vector delivery of siRNA and shRNA encoding genes into cultured and primary hematopoietic cells [M/OL]. In: Carmichael GG. ed. Methods in Molecular Biology, vol 309: RNA Silencing. New Jersey, USA: Humana Press, 2005. https://doi.org/10.1385/1-59259-935-4:261.
[16] 李跃萍, 宋丽萍, 邱曙东. 慢病毒载体在肿瘤基因治疗中的应用 [J]. 现代肿瘤医学, 2006, 14(12): 1614-1617.
[17] 陈志达, 丁丽华, 刘婕, 张亚楠, 朱杰, 罗晓丽, 刘国晓, 叶棋浓, 卫勃. 慢病毒载体介导E6AP基因敲低乳腺癌细胞株的构建及其功能检测 [J]. 军事医学, 2014, 38(11): 863-866,870.
[18] Swamy MN, Wu H, Shankar P. Recent advances in RNAi-based strategies for therapy and prevention of HIV-1/AIDS [J]. Adv Drug Deliv Rev, 2016, 103: 174-186.
[2] Kim DS, Nam JH. Characterization of attenuated coxsackievirus B3 strains and prospects of their application as live-attenuated vaccines [J]. Expert Opin Biol Ther, 2010, 10(2): 179-190.
[3] 汤晶晶, 张杰, 李凯, 樊帆, 李琼芬, 丁峥嵘.云南省2011—2014年急性弛缓性麻痹病例非脊髓灰质炎肠道病毒分子流行病学特征 [J]. 中国疫苗和免疫, 2017, 23(6): 608-612,646.
[4] Andréoletti L, Hober D, Becquart P, Belaich S, Copin MC, Lambert V, Wattré P. Experimental CVB3-induced chronic myocarditis in two murine strains: evidence of interrelationships between virus replication and myocardial damage in persistent cardiac infection [J]. J Med Virol, 1997, 52(2): 206-214.
[5] Lindberg AM, St?lhandske PO, Pettersson U. Genome of coxsackievirus B3 [J]. Virology, 1987, 156(1): 50-63.
[6] Kempf BJ, Barton DJ. Picornavirus RNA polyadenylation by 3Dpol, the viral RNA-dependent RNA polymerase [J]. Virus Res, 2015, 206: 3-11.
[7] Xu HT, Hassounah SA, Colby-Germinario SP, Oliveira M, Fogarty C, Quan Y, Han Y, Golubkov O, Ibanescu I, Brenner B, Stranix BR, Wainberg MA. Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors [J]. J Antimicrob Chemother, 2017, 72(3): 727-734.
[8] 张秀杰, 韩晓峰, 欧阳红生. HCV治疗的新方法:RNA依赖的RNA聚合酶抑制剂[J]. 生命的化学, 2007, 27(3): 251-253.
[9] Mondal M, Klimov P, Flynt AS. Rewired RNAi-mediated genome surveillance in house dust mites [J]. PLoS Genet, 2018, 14(1): e1007183.
[10] 王永佳, 陈永胜, 李国瑞. RNAi在植物中的作用机理及其应用研究进展[J]. 内蒙古民族大学学报(自然汉文版), 2010, 25(1): 54-58.
[11] MacKeigan JP, Murphy LO, Blenis J. Sensitized RNAi screen of human kinases and phosphatases identifies new regulators of apoptosis and chemoresistance [J]. Nat Cell Biol, 2005, 7(6): 591-600.
[12] Frank SB, Schulz VV, Miranti CK. A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vector [J]. BMC Biotechnol, 2017, 17(1): 24. doi: 10.1186/s12896-017-0341-x.
[13] Poling BC, Tsai K, Kang D, Ren L, Kennedy EM, Cullen BR. A lentiviral vector bearing a reverse intron demonstrates superior expression of both proteins and microRNAs [J]. RNA Biol, 2017, 14(11): 1570-1579.
[14] Rao DD, Vorhies JS, Senzer N, Nemunaitis J. siRNA vs. shRNA: similarities and differences [J]. Adv Drug Deliv Rev, 2009, 61(9): 746-759.
[15] Li M, Rossi JJ. Lentiviral vector delivery of siRNA and shRNA encoding genes into cultured and primary hematopoietic cells [M/OL]. In: Carmichael GG. ed. Methods in Molecular Biology, vol 309: RNA Silencing. New Jersey, USA: Humana Press, 2005. https://doi.org/10.1385/1-59259-935-4:261.
[16] 李跃萍, 宋丽萍, 邱曙东. 慢病毒载体在肿瘤基因治疗中的应用 [J]. 现代肿瘤医学, 2006, 14(12): 1614-1617.
[17] 陈志达, 丁丽华, 刘婕, 张亚楠, 朱杰, 罗晓丽, 刘国晓, 叶棋浓, 卫勃. 慢病毒载体介导E6AP基因敲低乳腺癌细胞株的构建及其功能检测 [J]. 军事医学, 2014, 38(11): 863-866,870.
[18] Swamy MN, Wu H, Shankar P. Recent advances in RNAi-based strategies for therapy and prevention of HIV-1/AIDS [J]. Adv Drug Deliv Rev, 2016, 103: 174-186.