本文選題：結(jié)核疫苗 + 腺相關(guān)病毒　； 參考：《華南理工大學(xué)》2011年碩士論文

【摘要】：結(jié)核病可累及全身多個臟器,其中肺結(jié)核最為常見。目前我國是世界上22個結(jié)核病高負(fù)擔(dān)國家之一,我國三分之一左右的人口已感染了結(jié)核桿菌,受感染人數(shù)超過4億。我國現(xiàn)有肺結(jié)核病人約500萬。如果不采取有效的控制措施,在未來的10年,我國可能有近5000萬的感染者發(fā)生結(jié)核病。對結(jié)核病的防治傳統(tǒng)的方法為接種卡介苗,但是缺陷大,反應(yīng)差異明顯,不能用于免疫缺陷的個體,同時為以后的檢測帶來諸多不便。研制更有效的結(jié)核疫苗具有深遠(yuǎn)的意義。 目的:以最新腺相關(guān)病毒載體系統(tǒng),構(gòu)建攜帶肺結(jié)核融合抗原(Ag85b-ESAT6)并整合siRNA(si-MCL-1)表達(dá)單位的二重表達(dá)新型結(jié)核核酸疫苗。包裝出含有目標(biāo)基因的重組腺相關(guān)病毒顆粒。分別構(gòu)建rAAV-EGFP與rAAV-siEGFP-EGFP腺相關(guān)病毒,包裝并通過病毒介導(dǎo)目標(biāo)基因在宿主細(xì)胞進(jìn)行表達(dá),驗證病毒載體表達(dá)水平。通過超濾濃縮純化rAAV病毒顆粒,提高病毒載體感染效率。方法:利用genetool軟件工具分析AAV-Helper-free系統(tǒng)中pAAV-MCS質(zhì)粒中的全部酶切位點,在不影響病毒包裝的前提下,確定酶切位點MluI作為siRNA表達(dá)框插入點。通過PCR方法擴(kuò)增融合抗原Ag85b-ESAT6與si-MCL-1(包括H1啟動子),si-EGFP(包括H1啟動子),將目的基因和表達(dá)框插入pAAV-MCS多克隆位點及相應(yīng)酶切位點,構(gòu)建并包裝rAAV-si-MCL-1-pVAE,rAAV-siEGFP-EGFP,rAAV-EGFP。其中pAAV-EGFP表達(dá)質(zhì)粒為實驗室已有資源。通過磷酸鈣共沉淀法將重組質(zhì)粒與包裝輔助質(zhì)粒pAAV-Helper,pAAV-RC共轉(zhuǎn)染AAV-293細(xì)胞制備重組病毒。重組病毒經(jīng)超濾濃縮,通過熒光顯微鏡以及流式細(xì)胞儀檢測表達(dá)效果。結(jié)果:重組子pAAV-si-MCL-1-pVAE ,pAAV-siEGFP-EGFP通過PCR鑒定和測序證實構(gòu)建成功。病毒感染HT1080在熒光顯微鏡和流式細(xì)胞儀中檢測到綠色熒光并存在差異,表明重組病毒包裝成功,siRNA表達(dá)單位正常工作不影響病毒包裝和蛋白表達(dá)。結(jié)論:成功包裝出具有侵染性和正常表達(dá)外源目的基因的重組腺相關(guān)病毒rAAV-si-MCL-1-pVAE,rAAV-siEGFP-EGFP,rAAV-EGFP。濃縮和純化后的rAAV病毒顆粒感染HT1080效果更明顯。本研究為研制優(yōu)于BCG的疫苗提供了新思路,具有深遠(yuǎn)的意義。
[Abstract]:Tuberculosis can involve multiple organs throughout the body, of which tuberculosis is the most common. At present, China is one of the 22 countries with high TB burden in the world. About 1/3 people in our country have been infected with Mycobacterium tuberculosis, and the number of infected people is more than 400 million. There are about 5 million tuberculosis patients in China. If effective control measures are not taken, there may be nearly 50 million cases of tuberculosis in China in the next 10 years. The traditional method to prevent and cure tuberculosis is to vaccinate BCG vaccine, but the defect is big, the reaction difference is obvious, can't be used in the individual of the immune deficiency, at the same time, bring many inconvenience for the later examination at the same time. It is of great significance to develop a more effective TB vaccine. Aim: to construct a novel recombinant tuberculosis nucleic acid vaccine carrying ag85b-ESAT6 and siRNA (si-MCL-1) expression unit by using the latest adeno-associated virus vector system. The recombinant adeno-associated virus particles containing the target gene were packaged. RAAV-EGFP and rAAV-siEGFP-EGFP adeno-associated viruses were constructed, and the target genes were expressed in host cells by virus mediated expression. RAAV virus particles were concentrated and purified by ultrafiltration to improve the efficiency of viral vector infection. Methods: the restriction sites of pAAV-MCS plasmid in AAV-Helper-free system were analyzed by genetool software, and the restriction site MluI was determined as the insertion point of siRNA expression frame without affecting virus packaging. The fusion antigen Ag85b-ESAT6 and si-MCL-1 (including H1 promoter) were amplified by PCR. The target gene and expression frame were inserted into the polyclonal site of pAAV-MCS and the corresponding restriction site, and the rAAV-siEGFP-EGFP pAAV-siEGFP-EGFP was constructed and packaged by PCR. Among them, pAAV-EGFP expression plasmid is a resource of laboratory. The recombinant virus was prepared by co-transfection of recombinant plasmid and pAAV-Helperon pAAV-RC into AAV-293 cells by calcium superphosphate coprecipitation. The recombinant virus was concentrated by ultrafiltration and detected by fluorescence microscope and flow cytometry. Results: the recombinant pAAV-si-MCL-1-pVAE pAAV-siEGFP-EGFP was successfully constructed by PCR and sequencing. Green fluorescence was detected by fluorescence microscope and flow cytometry in HT1080 infected by the virus, which indicated that the successful packaging of recombinant virus did not affect the viral packaging and protein expression. Conclusion: the recombinant adeno-associated virus rAAV-si-MCL-1-pVAEN-rAAV-siEGFP-EGFPN rAAV-EGFPhas been successfully packaged with infectious and normal expression of exogenous target gene. The concentrated and purified rAAV particles infected with HT1080 were more effective. This study provides a new idea for the development of a vaccine better than BCG, and has far-reaching significance.
【學(xué)位授予單位】：華南理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1

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