本文選題：基因治療 + 慢病毒載體��； 參考：《第一軍醫(yī)大學(xué)》2006年碩士論文

【摘要】：目的：以人類免疫缺陷病毒(HIV—1)為基礎(chǔ)構(gòu)建的慢病毒載體具有感染低分裂潛能細(xì)胞、整合目的基因至靶細(xì)胞基因組并可以長期穩(wěn)定表達(dá)、免疫反應(yīng)輕微等優(yōu)點，在臨床醫(yī)學(xué)和基礎(chǔ)醫(yī)學(xué)領(lǐng)域中有廣泛的應(yīng)用前景。 本系統(tǒng)采用“三質(zhì)粒+重組痘苗病毒”的方式生產(chǎn)慢病毒載體，期望獲得高拷貝數(shù)、安全型的適合臨床應(yīng)用的慢病毒載體。 方法：本系統(tǒng)主要生產(chǎn)方法為構(gòu)建主框架質(zhì)粒pVECRNA、包裝質(zhì)粒pGAGPOL及包膜質(zhì)粒pVSVG。通過脂質(zhì)體將這三個質(zhì)粒共轉(zhuǎn)染至BHK_(21)細(xì)胞，再用含有T_7RNA聚合酶基因的重組痘苗病毒vTF-3感染細(xì)胞，在生產(chǎn)細(xì)胞中，痘苗病毒轉(zhuǎn)錄翻譯系統(tǒng)指導(dǎo)T_7RNA聚合酶的轉(zhuǎn)錄和翻譯，然后T_7RNA聚合酶指導(dǎo)慢病毒載體cDNA的轉(zhuǎn)錄，痘苗病毒RNA聚合酶指導(dǎo)包裝蛋白p24等以及包膜蛋白水泡性口炎病毒包膜蛋白(VSV-G)的轉(zhuǎn)錄翻譯，最后由VSV-G包裝慢病毒載體RNA以及功能蛋白形成慢病毒顆粒，，經(jīng)細(xì)胞分泌釋放到培養(yǎng)上清中，收集培養(yǎng)上清經(jīng)0.22μm濾膜過濾得到慢病毒載體。 結(jié)果：RT-PCR及測序比對結(jié)果提示培養(yǎng)上清中含有慢病毒載體的基因組RNA。當(dāng)三質(zhì)粒與輔助痘苗病毒共轉(zhuǎn)染生產(chǎn)細(xì)胞BHK_(21)，48h后，共聚焦顯微鏡下觀察到生產(chǎn)細(xì)胞表達(dá)p24蛋白，并且其主要分布于胞質(zhì)中，而在細(xì)胞核中基本不表達(dá)，這提示質(zhì)粒pGAGPOL構(gòu)建成功；普通倒置熒光顯微鏡下觀察到生產(chǎn)細(xì)胞表達(dá)GFP，提示質(zhì)粒pVECRNA構(gòu)建成功，以上結(jié)果也提示生產(chǎn)系統(tǒng)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.
【學(xué)位授予單位】：第一軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2006
【分類號】：R392

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本文編號：1972515