摘要
目的建立一种基于三质粒的慢病毒包装细胞体系并对其感染效率进行检测。方法利用双酶切方法构建一个pLVTHM重组慢病毒表达载体，然后与pSPAX2，pMD2．G共转染HEK293T获取慢病毒原液，利用高速离心的方法对其进行浓缩纯化。设定不同感染复数（1，5，10，15，20），将上述制备的慢病毒制品感染HEK293T和HepG2肝癌细胞，荧光显微镜下观察绿色荧光蛋白表达水平以确认制备的病毒是否成功，其感染效率如何。结果利用一段非沉默性基因片段，构建了一个pLVTHM重组慢病毒表达栽体，将其与两个包装质粒共转染HEK293T细胞后，荧光显微镜下可见发出明亮绿色荧光，证明上述栽体成功转染到包装细胞中。于转染后42h和66h分剐收集细胞培养上清，获得含有慢病毒的原液，对病毒进行浓缩后获得浓度为l×10^8Tu／ml的病毒浓缩液。利用浓缩的病毒感染HEK293T和HepG2细胞，随着感染时间及感染复数值的增加，细胞内绿色荧光表达明显增强，感染后24h时HEK293T细胞内即可见明亮绿色荧光，其感染效率约为60％；在较难进行基因转染的HepG2细胞中，感染后24h后约40％的细胞可见明亮绿色荧光。结论成功建立了三质粒慢病毒包装细胞体系，该体系可成功表达含有目的基因的慢病毒，为后续系列研究提供了重要的工作平台。
Objective To establish a three-plasmid-based lentiviral packaging cell system and detect its efficiency. Methods A non-silencing pI.VTHM recombinant lentiviral expression vector was prepm-ed,then co-transfected with pSPAX2, pMD2. G into HEK293T cells, l.entiviral particals were concentrated and purified by high-speed centrifuge. The purified lentiviral products were actded to infect HEK293T and HepG2 cells,the level of expression of green fluorescent protein was observed by fluorescence mieroseopy in order to confirm the efficiency of the prepared lentivirus. Results Firstly constructed a recombinant lentiviral expression vector pLVTHM-shRNA by inserting a non-silence gene fragmenl into pLVTHM. After co-transfeetion with pLVTHM-shRNA ant] the packaging vector pSPAX2 and pMD2. G, HEK293T ceils expressed bright green fluorescence under a fluorescence microscope, proved that the vectors were successfully transfected into HEK293T cells. 42 and 66 hours after co-transfec.tion, supernatants containing lentivira｜ particals from HEK293T ceils were collected and concentrated. The concentration of the virus obtained was 1 x 10s Tl.l/ml. By increasing the infection time and virus volume,intracellular expression of green fluorescence were significantly enhanced,and bright green fluorescetlt was visible in HEK293T cells 24h after infection,the effi- ciency of infection was approximately 60% ;24 hours after infection,bright green fluorescenee was visible in about 40qc of HepG2 cells which were usually not easy to be transfered. Conclusion The three-plasmid lentiviral vector packaging system was successfully established and provided an importanl platfiorm for our fulure researches.
出处
《潍坊医学院学报》
2013年第1期1-3,F0003,共4页
Acta Academiae Medicinae Weifang
基金
国家自然科学基金(81272319)
山东省高等学校科技计划项目(J12LK02)
