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 293T cells for lend virus,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 interfer-ence 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 1×10^9IU/mL.The expression of ASIC3 mRNA and protein in PC12,BV2 and N2a 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.
作者
曲雪菲
华佳
吴进
龚爱华
蒋鹏
QU Xue-fei;HUA Jia;WU Jin;GONG Ai-hua;JIANG Peng(School of Medicine,Jiangsu University,Zhenjiang Jiangsu 212013;Department of Anesthesiology,Affiliated Hospital of Jiangsu University,Zhenjiang Jiangsu 212001,China)
出处
《江苏大学学报（医学版）》
CAS
2019年第1期58-61,66,共5页
Journal of Jiangsu University：Medicine Edition
基金
镇江社会发展科技支撑项目(SH2014078)
关键词
酸敏感离子通道3
干扰质粒
慢病毒
疼痛
acid-sensing ion channel 3(ASIC3)
interference plasmid
lentivirus
pain
