人hnRNPA2／B1shRNA慢病毒载体的构建  

Construction and Identification of Lentivirus Vector of shRNA for Human hnRNPA2/B1 Gene

作　　者：刘瑶[1] 石祥[2] 方文[2] 

机构地区：[1]贵州医科大学 [2]贵州医科大学临床生化教研室,贵州贵阳550004

出　　处：《贵阳医学院学报》2016年第6期630-634,共5页Journal of Guiyang Medical College

基　　金：国家自然科学基金(81560481)

摘　　要：目的:构建人核内不均一核糖核蛋白A2/B1(hnRNPA2/B1)基因的短发夹RNA(shRNA)慢病毒载体。方法:根据hnRNPA2/B1基因序列,设计4对hnRNPA2/B1 shRNA的干扰序列和1对阴性对照序列(NCShRNA);合成靶序列双链DNA,与p GMLV-SC5RNAi慢病毒载体连接,通过测序鉴定阳性克隆;用构建的慢病毒表达载体和包装质粒共转染HEK293T细胞,荧光显微镜下观察转染病毒72 h后细胞中绿色荧光蛋白的表达,以验证共转染是否成功,超滤法浓缩病毒原液并测定病毒滴度。结果:经酶切和BLAST对测序结果比对,重组克隆中插入的序列与设计合成的4对shRNA及1对阴性对照oligo DNA完全一致,提示慢病毒载体构建成功;荧光显微镜下,HEK 293T细胞可见强绿色荧光蛋白表达,慢病毒载体成功共转染入HEK 293T细胞,病毒滴度为5×1011TU/L。结论:成功构建人hnRNPA2/B1基因shRNA慢病毒载体,病毒滴度达5×1011TU/L。Objective： To construct lentivirus vector of shRNA for human hnRNPA2/B1 gene. Meth- ods： According to the sequence of hnRNPA2/B1 gene, 4 pairs of interference sequence containing hnRNPA2/B1 shRNA and 1 pairs of interference sequence containing negative control were designed. Targeted double chain DNA was synthesized and connected to the lentivirus vector. Positive clones were identified by sequencing. The constructed vector of slow virus expression vector and packaging plasmids were co-transfected to HEK293T cells. The expression of green fluorescent protein was ob- served under fluorescence microscope 72 h after virus transfection in order to verify whether co-trans- fection was successful or not. Uhrafihration concentrated virus solution and the virus titer was deter- mined. Results： Comparison of the results of enzyme digestion and blast sequencing showed that the insertion sequence of the recombinant clones was completely consistent with 4 pairs of shRNA and 1 pair negative control DNA oligo, indicating that the Lentivirus vector containing shRNA hnRNPA2/B1 was successfully constructed. Under the fluorescence microscope, the 293T HEK cells showed strong green fluorescent protein expression, indicating that the slow virus vector was successfully co-transfect- ed into 293T HEK cells. The virus titer was 5 × 10^11 tu/L. Conclusions： Lentivirus vector of shRNA for human hnRNPA2/B1 gene is successfully constructed, and the virus titer is 5 × 10^11 tu/L.

关 键 词：RNA干扰 慢病毒 293T细胞 载体 短发夹RNA 核内不均一核糖核蛋白基因 

分 类 号：Q782[生物学—分子生物学]