一种新型慢病毒载体制备体系的初步建立

发布时间：2018-06-03 10:49

本文选题：基因治疗 + 慢病毒载体　；参考：《第一军医大学》2006年硕士论文

【摘要】：目的：以人类免疫缺陷病毒(HIV—1)为基础构建的慢病毒载体具有感染低分裂潜能细胞、整合目的基因至靶细胞基因组并可以长期稳定表达、免疫反应轻微等优点，在临床医学和基础医学领域中有广泛的应用前景。本系统采用“三质粒+重组痘苗病毒”的方式生产慢病毒载体，期望获得高拷贝数、安全型的适合临床应用的慢病毒载体。方法：本系统主要生产方法为构建主框架质粒pVECRNA、包装质粒pGAGPOL及包膜质粒pVSVG。通过脂质体将这三个质粒共转染至BHK_(21)细胞，再用含有T_7RNA聚合酶基因的重组痘苗病毒vTF-3感染细胞，在生产细胞中，痘苗病毒转录翻译系统指导T_7RNA聚合酶的转录和翻译，然后T_7RNA聚合酶指导慢病毒载体cDNA的转录，痘苗病毒RNA聚合酶指导包装蛋白p24等以及包膜蛋白水泡性口炎病毒包膜蛋白(VSV-G)的转录翻译，最后由VSV-G包装慢病毒载体RNA以及功能蛋白形成慢病毒颗粒，，经细胞分泌释放到培养上清中，收集培养上清经0.22μm滤膜过滤得到慢病毒载体。结果：RT-PCR及测序比对结果提示培养上清中含有慢病毒载体的基因组RNA。当三质粒与辅助痘苗病毒共转染生产细胞BHK_(21)，48h后，共聚焦显微镜下观察到生产细胞表达p24蛋白，并且其主要分布于胞质中，而在细胞核中基本不表达，这提示质粒pGAGPOL构建成功；普通倒置荧光显微镜下观察到生产细胞表达GFP，提示质粒pVECRNA构建成功，以上结果也提示生产系统正常工作。
[Abstract]:Objective: the lentivirus vector constructed on the basis of human immunodeficiency virus (HIV-1) has the advantages of infecting low mitotic potential cells, integrating the target gene into the target cell genome and expressing it stably for a long time. It is widely used in clinical medicine and basic medicine. The lentivirus vector was produced by "three plasmids recombinant vaccinia virus" in order to obtain high copy number and safety lentivirus vector suitable for clinical application. Methods: the main production method of the system was to construct the main frame plasmid pVECRNAs, package plasmid pGAGPOL and encapsulated plasmid pVSVG. The three plasmids were co-transfected into BHKS21 cells by liposome, and then the recombinant vaccinia virus vTF-3 containing T_7RNA polymerase gene was used to infect the cells. In the production cells, the vaccinia virus transcription translation system directed the transcription and translation of T_7RNA polymerase. Then T_7RNA polymerase directed the transcription of lentivirus vector cDNA, vaccinia virus RNA polymerase directed the transcriptional translation of packaging protein p24 and encapsulated protein vesicular stomatitis virus envelope protein (VSV-GV). Finally, lentivirus particles were formed by packaging lentivirus vector RNA and functional proteins by VSV-G, and then released into culture supernatant by cell secretion. Lentivirus vector was obtained by 0.22 渭 m filtration of culture supernatant. Results the results of RT-PCR and sequencing indicated that the genomic RNAs containing lentivirus vectors were found in the supernatants. When the three plasmids were co-transfected with the adjuvant vaccinia virus into the production cell BHKS for 48 h, the expression of p24 protein was observed under confocal microscope, and the p24 protein was mainly distributed in the cytoplasm, but not expressed in the nucleus. This indicated that the plasmid pGAGPOL was successfully constructed. The expression of GFP in the production cells was observed under the general inverted fluorescence microscope, indicating that the plasmid pVECRNA was successfully constructed, and the above results also indicated that the production system was working normally.
【学位授予单位】：第一军医大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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