ASIC3-shRNA干扰慢病毒质粒的构建及其效率的鉴定
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摘要
目的:构建酸敏感离子通道3(acid-sensing ion channels 3,ASIC3)干扰慢病毒质粒并进行效率鉴定。方法:利用Gen Bank获取ASIC3基因序列并合成相应干扰序列,通过T4DNA连接酶将ASIC3基因片段连接至环状Plko. 1-Puro载体,将重组质粒转染293T细胞获取慢病毒,用实时荧光定量PCR(qRT-PCR)检测病毒滴度;将包装之后的慢病毒感染PC12、BV2、N2a细胞,分别分为ASIC3-shRNA组和EGFP-shRNA组(阴性对照),经慢病毒感染后用qRT-PCR和免疫印迹技术分别检测ASIC3 mRNA和蛋白表达。结果:合成的ASIC3干扰序列成功连接至Plko. 1-Puro载体; qRT-PCR及DNA测序鉴定结果证实,ASIC3-shRNA质粒构建成功; qRT-PCR与免疫印迹结果表明,在PC12、BV2、N2a细胞中,与EGFP-shRNA组相比,ASIC3-shRNA组ASIC3 mRNA和蛋白表达量均明显下调(P <0. 05)。病毒滴度约为1×109IU/mL。结论:ASIC3-shRNA干扰慢病毒质粒构建成功。
Objective: To construct lentiviral interference plasmid of acid-sensing ion channel 3( ASIC3) and validate the efficiency. Methods: ASIC3 gene sequence was obtained from Genbank to synthesize the corresponding interference sequence. Then it was inserted into the circular Plko. 1-Puro vector by T4 DNA ligase. The recombinant plasmid was transfected into 293 T cells for lentivirus,and the obtained lentivirus was determined for titer by quantitative real-time PCR( qRT-PCR). PC12,BV2 and N2 a cells were infected with the packaging lentivirus,and divided into ASIC3-shRNA group and EGFPshRNA group( negative control). After lentivirus infection,qRT-PCR and western blotting were used to detect the expression of ASIC3 mRNA and protein,respectively. Results: The synthetic ASIC3 interference sequence was successfully inserted into Plko. 1-Puro vector. qRT-PCR and DNA testing confirmed the successful construction of the ASIC3-shRNA plasmid,the virus titer was approximately 1 × 109 IU/mL. The expression of ASIC3 mRNA and protein in PC12,BV2 and N2 a cells were significantly downregulated in ASIC3-shRNA group compared with EGFP-shRNA group( P < 0. 05). Conclusion: The ASIC3-shRNA interference lentivirus plasmid was successfully constructed.
Objective: To construct lentiviral interference plasmid of acid-sensing ion channel 3( ASIC3) and validate the efficiency. Methods: ASIC3 gene sequence was obtained from Genbank to synthesize the corresponding interference sequence. Then it was inserted into the circular Plko. 1-Puro vector by T4 DNA ligase. The recombinant plasmid was transfected into 293 T cells for lentivirus,and the obtained lentivirus was determined for titer by quantitative real-time PCR( qRT-PCR). PC12,BV2 and N2 a cells were infected with the packaging lentivirus,and divided into ASIC3-shRNA group and EGFPshRNA group( negative control). After lentivirus infection,qRT-PCR and western blotting were used to detect the expression of ASIC3 mRNA and protein,respectively. Results: The synthetic ASIC3 interference sequence was successfully inserted into Plko. 1-Puro vector. qRT-PCR and DNA testing confirmed the successful construction of the ASIC3-shRNA plasmid,the virus titer was approximately 1 × 109 IU/mL. The expression of ASIC3 mRNA and protein in PC12,BV2 and N2 a cells were significantly downregulated in ASIC3-shRNA group compared with EGFP-shRNA group( P < 0. 05). Conclusion: The ASIC3-shRNA interference lentivirus plasmid was successfully constructed.
引文
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[9]Herrera-Carrillo E,Liu YP,Berkhout B.Improving miRNA delivery by optimizing miRNA expression cassettes in diverse virus vectors[J].Hum Gene Ther Methods,2017,28(4):177-190.
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[13]Wu P,Wilmarth MA,Zhang F,et al.miRNA and shR-NA expression vectors based on mRNA and miRNA processing[J].Methods Mol Biol,2013,936:195-207.
[14]Lambeth LS,Smith CA.Short hairpin RNA-mediated gene silencing[J].Methods Mol Biol,2013,942:205-232.
[15]ter Brake O,Westerink JT,Berkhout B.Lentiviral vector engineering for anti-HIV RNAi gene therapy[J].Methods Mol Biol,2010,614:201-213.
[2]Yen YT,Tu PH,Chen CJ,et al.Role of acid-sensing ion channel 3 in sub-acute-phase inflammation[J].Mol Pain,2009,5:1.
[3]Ni L,Fang P,Hu ZL,et al.Identification and function of acid-sensing ion channels in RAW 264.7 macrophage cells[J].Curr Med Sci,2018,38(3):436-442.
[4]Deval E,Gasull X,Noёl J,et al.Acid-sensing ion channels(ASICs):pharmacology and implication in pain[J].Pharmacol Ther,2010,128(3):549-558.
[5]Deval E,Noёl J,Lay N,et al.ASIC3,a sensor of acidic and primary inflammatory pain[J].EMBO J,2008,27(22):3047-3055.
[6]Hiasa M,Okui T,Allette YM,et al.Bone pain induced by multiple myeloma is reduced by targeting V-ATPase and ASIC3[J].Cancer Res,2017,77(6):1283-1295.
[7]Izurieta Munoz H,Gonzales EB,Sumien N.Effects of creatine supplementation on nociception in young male and female mice[J].Pharmacol Rep,2017,70(2):316-321.
[8]Ren P,Wang WB,Pan HH,et al.Up-regulation of ASIC3 expression byβ-estradiol[J].Neurosci Lett,2018,684:200-204.
[9]Herrera-Carrillo E,Liu YP,Berkhout B.Improving miRNA delivery by optimizing miRNA expression cassettes in diverse virus vectors[J].Hum Gene Ther Methods,2017,28(4):177-190.
[10]Liu YP,Berkhout B.miRNA cassettes in viral vectors:problems and solutions[J].Biochim Biophys Acta,2011,1809(11/12):732-745.
[11]孟凡荣,陈琛,万海粟,等.慢病毒载体及其研究进展[J].中国肺癌杂志,2014,17(12):870-876.
[12]Sioud M.RNA interference:mechanisms,technical challenges,and therapeutic opportunities[J].Methods Mol Biol,2015,1218:1-15.
[13]Wu P,Wilmarth MA,Zhang F,et al.miRNA and shR-NA expression vectors based on mRNA and miRNA processing[J].Methods Mol Biol,2013,936:195-207.
[14]Lambeth LS,Smith CA.Short hairpin RNA-mediated gene silencing[J].Methods Mol Biol,2013,942:205-232.
[15]ter Brake O,Westerink JT,Berkhout B.Lentiviral vector engineering for anti-HIV RNAi gene therapy[J].Methods Mol Biol,2010,614:201-213.