贵州医科大学学报

2017, v.42;No.201(06) 621-625

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人Spsb1基因慢病毒干扰载体的构建及病毒包装
Construction of Lentivirus shRNA Interference Vector of Human Spsb1 Gene and its Lentivirus Particle Packaging

孙达权;石松;徐莹莹;雷霆雯;莫晓川;王筑婷;李红梅;徐国强;
SUN Daquan;SHI Song;XU Yingying;LEI Tingwen;MO Xiaochuan;WANG Zhuting;LI Hongmei;XU Guoqiang;School of Basic Medine,Guizhou Medical University;

摘要(Abstract):

目的:构建人Spsb1基因的shRNA慢病毒干扰载体,为研究该基因在肝癌细胞生长、增殖、转移和浸润中的作用打下前期基础。方法:根据shRNA的设计原则设计一段shRNA和阴性对照shRNA,化学合成DNA双链中的两条单链,通过退火和配对后与慢病毒干扰载体LV-3(p GLVH1/GFP+Puro)连接,阳性克隆子LV3-Spsb1测序鉴定;将LV3-Spsb1和辅助质粒p CMV-delta8.91/p CMV-VSVG共转染HEK293T细胞,转染后24 h在荧光显微镜下观察细胞转染效率;用转染后收集到的病毒液感染肝癌细胞SMMC-7721,荧光显微镜下观察细胞荧光;用1 mg/L嘌呤霉素杀死未感染的细胞,Western blot检测细胞内源性Spsb1基因表达量。结果:测序鉴定证明获得的LV3-Spsb1质粒是所需要的目的慢病毒shRNA干扰载体;通过与辅助质粒共转染HEK293T细胞后,细胞在荧光显微镜下发出明亮的绿色荧光,提示细胞转染效率较高;病毒液感染SMMC-7721后,细胞内出现绿色荧光,证明共转染系统可以成功包装shRNA-Spsb1重组慢病毒颗粒;Western blot结果显示shRNA-Spsb1重组慢病毒能够抑制细胞内Spsb1基因表达,证明shRNA-Spsb1的确可以降低肝癌细胞内源性Spsb1基因表达,可用于后期的基因功能研究。结论:成功构建人Spsb1基因的慢病毒shRNA干扰载体LV3-Spsb1并包装出其慢病毒颗粒。
Objective: To construct lentivirus shRNA interference vector of human Spsb1( splA/ryanodine receptor domain and SOCS box containing 1) gene and to lay a foundation for uncovering the function of human Spsb1 gene in hepatocellular carcinoma development and metastasis. Methods:Firstly,a fragment of shRNA sequence for Spsb1 and a negative control shRNA were designed according to the shRNA design principles; Secondly,two single DNA strands of shRNAs,including shRNA of Spsb1 and the control,were synthesized by gene corporation,and then were ligated with lentivirus interference vector named LV3( p GLVH1/GFP + Puro) after their annealing and renaturation. Positive clone LV3-Spsb1 was verified by DNA sequencing; Thirdly,co-transfection was implemented to mediate expressional plasmid of LV3-Spsb1 and auxiliary plasmids of p CMV-delta8. 91 and p CMV-VSVG into HEK293 T cells. In 24 h after transfection,the transfection efficiency was confirmed with fluorescent microscopy; Fourthly,virus solution was collected by centrifugation under low temperature for 5 min at3000 g and filtrating by hydrophilic filter membrane in 48 h after transfection. The collected virus solution was then stored in-80 ℃ after subpackage; Fifthly,virus solution was added into hepatocellular carcinoma cell SMMC-7721 which was detected by fluorescent microscope in 72 h after infection; Lastly,western blot was used to detected the expression of SPSB1 protein in infected SMMC-7721 cells which survived in 1 mg/L puromycin screening culture medium for a week. Results: DNA sequencing showed that positive clone LV3-Spsb1 was just the goal lentivirus shRNA interference vector; HEK293 T cells,co-transfected by lentivirus expressional vector LV3-Spsb1 and auxiliary vectors p CMV-delta8. 91 and p CMV-VSVG,showed bright green fluorescence under fluorescent microscope which proved the transfection efficiency; SMMC-7721 cells showed green fluorescence after infection by viral particles which verified that shRNA-Spsb1. Lentivirus particle package was successful; western blot results showed the expression of SPSB1 protein decreased in infected SMMC-7721 cells which indicated that lentivirus vector LV3-Spsb1 could knock down the gene expression of Spsb1. Conclusion: Lentivirus shRNA expressional vector LV3-Spsb1 was successfully constructed and its lentivirus particle was packaged.

关键词(KeyWords): 肝肿瘤;慢病毒干扰载体;脂质体;转染;Spsb1基因
liver neoplasms;lentiviral interference vector;liposomes;transfection;Spsb1 gene

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81560390);; 贵州省科学技术基金[黔科合J字(2010)2,黔科合J字(2014)2025号];; 贵阳医学院院基金(院基金合同字第017号)

作者(Author): 孙达权;石松;徐莹莹;雷霆雯;莫晓川;王筑婷;李红梅;徐国强;
SUN Daquan;SHI Song;XU Yingying;LEI Tingwen;MO Xiaochuan;WANG Zhuting;LI Hongmei;XU Guoqiang;School of Basic Medine,Guizhou Medical University;

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DOI: 10.19367/j.cnki.1000-2707.2017.06.002

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