SP110基因过表达慢病毒载体的构建与包装  

Construction and packaging of SP110 gene overexpressed lentivirus vector

作　　者：贾鹏霞 刘丙雨 李新月 张万江[1] 程江[1,2] 吴江东[1] 马雅静[1,2] JIA Pengxia;LIU Bingyu;LI Xinyue;ZHANG Wanjiang;CHENG Jiang;WU Jiangdong;MA Yajing(Key Laboratory of Xinjiang Endemic and Ethnic Diseases,Shihezi University,Shihezi Xinjiang 832000,China;Department of Clinical Laboratory,the First Affiliated Hospital of Shihezi University School of Medicine,Shihezi Xinjiang 832000,China)

机构地区：[1]石河子大学新疆地方病与民族高发病教育部重点实验室,新疆石河子832000 [2]石河子大学医学院第一附属医院检验科,新疆石河子832000

出　　处：《转化医学杂志》2020年第2期75-78,共4页Translational Medicine Journal

基　　金：国家自然科学基金委员会-新疆联合基金资助项目(U1803127);兵团重点领域科技攻关计划项目(2018AB019)。

摘　　要：目的构建核体蛋白SP110基因过表达慢病毒载体,并进行慢病毒包装,为研究SP110在结核病中的作用机制奠定基础。方法利用PCR技术获得SP110基因序列并将其插入到慢病毒穿梭质粒LV5中,获得重组慢病毒质粒V-SP110,经酶切鉴定及DNA测序鉴定后,用RNAi-Mate将重组质粒和慢病毒包装质粒系统(pGag/Pol、pRev、pVSV-G)包装成重组慢病毒颗粒,共转染293T细胞,包装产生慢病毒。通过荧光显微镜或FACS计数荧光细胞,结合稀释倍数计算病毒滴度。结果酶切鉴定结果显示产生约1650 bp的片段,片段大小与SP110基因cDNA大小一致。DNA测序比对测序结果与预期SP110基因序列完全一致,说明SP110基因正确插入载体中,成功构建了SP110基因过表达载体。经293T细胞包装后,成功获得病毒滴度为1.0×10^8 TU/mL的重组慢病毒LV5-SP110。结论SP110基因过表达慢病毒载体构建与包装成功完成,为进一步探讨SP110基因在结核病发生发展中的作用提供工具。Objective To construct and package the nucleosomal protein SP110 gene overexpression lentivirus vector,laying foundation for studying the mechanism of SP110 in tuberculosis.Methods The SP110 gene was inserted into plasmid LV5 of lentiviral vector by recombinant DNA technology.Lentivirus vector V-SP110 was got after recombination.The combinant lentivirus vector was detected by restriction endonuclease digestion and DNA sequencing.The recombinant lentiviral plasmid V-SP110 was transfected into 293T cells with the packaging plasmids including pGag/Pol,pRev and pVSV-G by RNAi-Mate.The recombinant lentivirus expressing SP110 was obtained from the cells supernatant and the viral titer was calculated by flow cytometry and dilution factor.Results The results of recombinant plasmids digestion identification showed that the fragment is about 1650 bp,which is the same size as SP110 gene cDNA fragments.The blast results of SP110 cDNA sequence showed that the sequencing results and expected SP110 gene sequence is completely consistent.It was confirmed that the SP110 was correctly inserted into the vector,and that SP110-gene-over-expressed lentivirus vectors was successfully constructed.After packing 293T cells,we successful got recombinant lentivirus LV5-SP110 with virus drops to 1.0×10^8 TU/mL.Conclusion The construction and packaging of the lentivirus vector overexpressing SP110 gene were successfully completed,providing a tool for further exploring the role of SP110 gene in the occurrence and development of tuberculosis.

关 键 词：SP110基因 慢病毒构建 慢病毒包装 

分 类 号：R349.6[医药卫生—基础医学]