β-arrestin 2抗基因RNA慢病毒表达载体的构建与鉴定  

Construction and Identification of Lentivector Encoding beta arrestin 2 Antigene RNAs

作　　者：卜慧莲[1] 温晓岩[2] 刘希江[1] 徐懋[3] 曹菲[1] 田学愎[1] 杨辉[1] 高峰[1] 田玉科[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院麻醉学教研室,武汉430030 [2]山东省聊城市人民医院检验科,聊城252000 [3]北京大学第三医院麻醉科,北京100191

出　　处：《华中科技大学学报（医学版）》2011年第1期37-40,46,共5页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金资助项目(No.30700783);高等学校博士学科点专项科研基金新教师项目(No.20070487117);贝朗麻醉科学研究基金(2007)资助项目

摘　　要：目的构建携带β-arrestin 2抗基因RNA的慢病毒表达载体并对其进行鉴定。方法将线性化的慢病毒载体pGC-FU与抗基因RNA在In-Fusion交换酶作用下构建目的质粒pGC-agRNA,转化感受态细胞,对长出的克隆应用菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和比对分析。重组病毒质粒与另外两种辅助包装原件载体质粒通过LipofectamineTM2000共转染293T细胞,培养48 h后,收集细胞培养上清液,将病毒浓缩后在293T细胞中测定病毒滴度,并检测慢病毒载体在工具细胞NG108-15细胞的转染效率。结果抗基因RNA被成功构建入慢病毒表达载体pGC-FU,病毒滴度为2×109TU/mL。用该慢病毒感染NG108-15细胞,当感染复数(MOI)为100时,感染效率大于95%。结论成功构建了携带β-arrestin 2抗基因RNA的慢病毒表达载体,为研究β-arrestin 2在μ阿片受体调控机制中的作用提供了有效的研究工具,而且为抗基因RNA技术应用于体内外实验研究奠定了基础。Objective To construct lentivector encoding antigene RNAs（agRNAs）targeting beta arrestin 2 gene（Arrb2）and identify its titer and infection efficiency.Methods The linearized lentiviral vector pGC-FU and agRNAs were constructed into aim plasmid pGC-agRNA under the effect of In-Fusion convertase.The aim plasmids were transformed into competent cells.The grown colonies were identified by colony PCR,and then the positive colonies were sequenced and aligned.Recombinant lentivector plasmids and the other two helping plasmids were co-transfected into 293T cells by LipofectamineTM2000,and cell culture supernatant was collected after 48 h.The virus supernatant was concentrated and titered in 293T cells.The infection efficiency of the constructed virus was determined in NG108-15 cells.Results β-arrestin 2 agRNAs were successfully cloned into lentivector pGC-FU.The titer of concentrated virus was 2×109 TU/mL.The infection efficiency of the virus in NG108-15 cells was more than 95%,when the MOI was 100.Conclusion Lentivector encoding agRNAs has been successfully constructed,providing an effective tool for researching the role of beta arrestin 2 gene in the regulation of μ opioid receptor and laying foundation for application of antigene technique in vivo and in vitro experiments.

关 键 词：基因沉默 抗基因RNA 慢病毒 β-arrestin2 

分 类 号：Q782[生物学—分子生物学]