本文選題：核酸疫苗 + 肝臟特異性��； 參考：《華南理工大學(xué)》2012年碩士論文

【摘要】：據(jù)中國衛(wèi)生部2010年最新統(tǒng)計，目前中國仍約有9300萬人為無癥狀乙肝病毒攜帶者，乙型肝炎病毒的傳染性比艾滋病毒強50至100倍，每年發(fā)病患者數(shù)在百萬以上。乙肝免疫耐受是導(dǎo)致乙肝治療失敗的主要原因。臨床治療乙肝的藥物存在高耐藥、低耐受、不良反應(yīng)等問題。傳統(tǒng)的第二代疫苗本身的一些缺點（如部分接種者無免疫應(yīng)答，出現(xiàn)副反應(yīng)，對乙肝患者無效），及病毒本身不閉合等特點使得兼有預(yù)防和治療作用的核酸疫苗成為熱點。最新采用的RNAi技術(shù)治療乙肝，雖然取得了一定的研究進展，但其組織特異性差，表達(dá)持續(xù)性弱，載體投遞效率低，使得藥物開發(fā)面臨瓶頸。 本研究采用肝臟特異性的二類啟動子hAAT構(gòu)建靶向HBV HBsAg基因的肝臟特異性siRNA表達(dá)單元，結(jié)合人工優(yōu)選HBV表位構(gòu)建多表位抗原基因HB，最終構(gòu)建了AAV介導(dǎo)的特異性表達(dá)siRNA的新型乙肝多表位疫苗，為打破乙肝免疫耐受、消除基于RNAi技術(shù)藥物的副作用以及提高疫苗的投遞效率打下堅實的基礎(chǔ)。 首先通過重組PCR將肝臟特異性啟動子hAAT、終止子U13’box、兩個相同的增強子APOE及siHBsAg基因拼接構(gòu)建成為肝臟特異性siHBsAg表達(dá)單元，將表達(dá)單元插入AAV-MCS質(zhì)粒中，構(gòu)建重組質(zhì)粒pAAV-siHBsAg，ELISA檢測siRNA的抑制效果。然后將HBV多表位抗原基因HB融合EGFP的蛋白基因通過分子克隆技術(shù)構(gòu)建在pAAV-siHBsAg質(zhì)粒中,得到pAAV-siHBsAg-HE，將重組質(zhì)粒pAAV-siHBsAg-HE與包裝質(zhì)粒pAAV-RC和輔助質(zhì)粒pHelper用磷酸鈣法共轉(zhuǎn)染AAV-293細(xì)胞制備重組病毒rAAV-siHBsAg-HE，并通過陰陽離子交換及超濾離心等純化濃縮。構(gòu)建的病毒rAAV-siHBsAg-HE感染HT1080細(xì)胞，通過觀察綠色熒光，驗證病毒的組裝效果及HB-EGFP基因在AAV病毒載體中的表達(dá)，并且通過流式細(xì)胞術(shù)檢測報告基因EGFP測定病毒滴度。再次使用ELISA檢測基于腺相關(guān)病毒載體siRNA的抑制效果。最終結(jié)果是成功地構(gòu)建了重組質(zhì)粒pAAV-siHBsAg和pAAV-siHBsAg-HE。EGFP綠色熒光觀察顯示HB在rAAV中可以有效的表達(dá)。通過蛋白電泳檢測及病毒電鏡圖片顯示最終得到高純度、高濃縮、高滴度的AAV病毒。ELISA檢測表明構(gòu)建的特異性表達(dá)siRNA的質(zhì)粒和AAV病毒中均有抑制效果；AAV介導(dǎo)的特異性表達(dá)siRNA的新型乙肝多表位疫苗的成功構(gòu)建和驗證為進一步研究乙肝核酸疫苗提供了實驗基礎(chǔ)。
[Abstract]:According to the latest statistics from China's Ministry of Health in 2010, there are still about 93 million asymptomatic hepatitis B virus carriers in China. Hepatitis B virus is 50 to 100 times more infectious than HIV.Hepatitis B immune tolerance is the main cause of failure of hepatitis B treatment.Clinical treatment of hepatitis B drugs have high drug resistance, low tolerance, adverse reactions and other problems.Some disadvantages of the traditional second-generation vaccines (such as no immune response of some vaccinators, side effects, ineffective to hepatitis B patients, and the virus itself are not closed) make the nucleic acid vaccine with both preventive and therapeutic functions a hot spot.Although the latest RNAi technology has made some progress in the treatment of hepatitis B, it has poor tissue specificity, weak expression persistence and low delivery efficiency, which makes the drug development face the bottleneck.In this study, liver-specific siRNA expression units targeting HBV HBsAg gene were constructed by liver-specific second class promoter hAAT.In order to break the immune tolerance of hepatitis B virus, a novel hepatitis B polyepitope vaccine mediated by AAV was constructed by combining with the artificially selected HBV epitope gene HBs, and a novel hepatitis B polyepitope vaccine with specific expression of siRNA was constructed.Eliminating the side effects of drugs based on RNAi technology and improving the delivery efficiency of vaccines lay a solid foundation.Firstly, liver specific promoter hat, Terminator U13box, two identical enhancers APOE and siHBsAg genes were spliced into liver-specific siHBsAg expression units by recombinant PCR. The expression units were inserted into AAV-MCS plasmids.The inhibitory effect of recombinant plasmid pAAV-sibsAg on siRNA was detected by Elisa.Then the HBV polyepitope gene HB fused with EGFP protein gene was constructed into the pAAV-siHBsAg plasmid by molecular cloning.The recombinant plasmid pAAV-siHBsAg-HE, package plasmid pAAV-RC and auxiliary plasmid pHelper were co-transfected into AAV-293 cells by calcium phosphate method to prepare the recombinant virus rAAV-sibsAg-HEE. The recombinant virus was purified and concentrated by anion exchange and ultrafiltration centrifugation.The constructed virus rAAV-siHBsAg-HE was infected with HT1080 cells. By observing the green fluorescence, the viral assembly effect and the expression of HB-EGFP gene in the AAV virus vector were verified. The titer of the reporter gene EGFP was detected by flow cytometry.ELISA was used again to detect the inhibitory effect of adeno-associated virus vector siRNA.The final result was that the recombinant plasmid pAAV-siHBsAg and pAAV-siHBsAg-HE.EGFP were successfully constructed. The results showed that HB could be expressed effectively in rAAV.High purity and concentration were finally obtained by protein electrophoresis and electron microscope images of the virus.High titer detection of AAV virus. Elisa showed that the constructed plasmids expressing siRNA and AAV virus had inhibitory effect. The successful construction and validation of a novel hepatitis B multiepitope vaccine with specific siRNA expression mediated by AAV could be further studied.Liver nucleic acid vaccine provides experimental basis.
【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】：2012
【分類號】：R392

【參考文獻】

相關(guān)期刊論文 前9條

1 縱書芳;陳勇;王莉娟;劉興祥;徐云芳;;靶向乙型肝炎病毒X區(qū)的siRNAs對HepG2.2.15細(xì)胞HBV復(fù)制的抑制作用[J];實用肝臟病雜志;2010年04期

2 張利濤;劉慧琳;曹以誠;;肝臟特異性表達(dá)載體的構(gòu)建及Western blotting檢測[J];中國畜牧獸醫(yī);2011年01期

3 陶嫦立;曹以誠;;乙型肝炎多表位DNA疫苗的發(fā)展趨勢[J];生命的化學(xué);2006年01期

4 黃鏡賢;曹以誠;杜正平;陶嫦立;楊化強;;共表達(dá)siRNA和hIL-12的新型乙肝多表位DNA疫苗的研究[J];中國生物工程雜志;2008年08期

5 方翔;杜正平;曹以誠;郭旭;劉鵬飛;董守斌;;siRNA pro 2.0:siRNA理性設(shè)計在線程序[J];中國生物化學(xué)與分子生物學(xué)報;2007年09期

6 李楊;王賽鋒;王亞男;孟頌東;;乙肝治療性疫苗的研制與臨床應(yīng)用[J];生物技術(shù)通報;2010年04期

7 薛皓;張穎倩;薛付忠;馬春紅;;乙型肝炎病毒多表位疫苗的研究進展及現(xiàn)有的常用表位[J];預(yù)防醫(yī)學(xué)論壇;2008年12期

8 洪源;成軍;;RNA干擾與慢性乙型肝炎治療[J];中華實驗和臨床感染病雜志(電子版);2007年04期

9 ;Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency[J];World Journal of Gastroenterology;2007年44期

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