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GenBank获取ASIC3基因序列并合成相应干扰序列，通过T4 DNA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1×109 IU/mL。结论： ASIC3shRNA干扰慢病毒质粒构建成功。

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关键词 ：酸敏感离子通道3, 干扰质粒, 慢病毒, 疼痛

Abstract：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 293T cells for lentivirus, and the obtained lentivirus was determined for titer by quantitative realtime PCR(qRTPCR). PC12, BV2 and N2a cells were infected with the packaging lentivirus, and divided into ASIC3shRNA group and EGFPshRNA 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 N2a cells were significantly downregulated in ASIC3shRNA group compared with EGFPshRNA group(P<0.05). Conclusion: The ASIC3shRNA interference lentivirus plasmid was successfully constructed.

基金资助:镇江社会发展科技支撑项目(SH2014078)

通讯作者: 蒋鹏(通讯作者)，主任医师，E-mail: doctorjp@163.com

作者简介: 曲雪菲(1992—)，女，硕士研究生

引用本文:

曲雪菲，华佳，吴进，龚爱华，蒋鹏. ASIC3-shRNA干扰慢病毒质粒的构建及其效率的鉴定[J]. 江苏大学学报:医学版, 2019, 29(01): 58-61. QU Xue-fei1,2, HUA Jia2, WU Jin2, GONG Ai-hua1, JIANG Peng2. Construction of ASIC3shRNA interference lentiviral plasmid and its efficiency validation. Journal of Jiangsu University(Medicine Edition), 2019, 29(01): 58-61.

链接本文:

http://zzs.ujs.edu.cn/xbyxb/CN/ 或 http://zzs.ujs.edu.cn/xbyxb/CN/Y2019/V29/I01/58

［1］Zhu H, Ding J, Wu J, et al. Resveratrol attenuates bone cancer pain through regulating the expression levels of ASIC3 and activating cell autophagy\[J\]. Acta Biochim Biophys Sin (Shanghai), 2017，49（11）：1008-1014.

［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 by β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 shRNA 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.