本文選題：基于 + 細胞　； 參考：《中國科學(xué)技術(shù)大學(xué)》2006年博士論文

【摘要】：盡管預(yù)防性亞單位重組乙肝疫苗和一些治療性藥物在臨床上已經(jīng)廣泛的應(yīng)用，乙肝病毒(Hepatitis B Virus，HBV)感染目前仍然是嚴重一個威脅人類健康的尚未攻克的疾病。由于DNA疫苗具有誘導(dǎo)體液免疫和細胞免疫的獨特的能力，因此為預(yù)防和治療HBV感染提供了一個新的發(fā)展方向。 近年來進行的Ⅰ期臨床實驗結(jié)果證明HBV DNA疫苗可以在健康人群中誘導(dǎo)保護性反應(yīng)：在慢性HBV感染人群中可以誘導(dǎo)特異性的T細胞反應(yīng)和清除HBVDNA。然而，這些臨床實驗中使用的HBV DNA疫苗只編碼單一的抗原蛋白——S抗原或PreS2/S抗原。而且由于DNA疫苗獨特的抗原表達和遞呈途徑，它的免疫原性仍然需要進一步的改進。因此，我們根據(jù)HBV表面抗原(HBsAg)上的T細胞表位，設(shè)計了一個包含多個細胞毒性T細胞(CTL)表位和輔助性T細胞(Th)表位的多表位DNA疫苗，并研究了它的功能，同時研究了結(jié)核桿菌熱休克蛋白(Heat Shock Protein，HSP)HSP70的分子佐劑功能。 結(jié)果表明，在H-2~d和H-2~b小鼠模型中，多表位HBV DNA疫苗誘導(dǎo)了一個比單一抗原基因的HBsAg DNA疫苗更強的HBsAg特異性CTL反應(yīng)。然而，在H-2~d小鼠模型中，D~d-限制性表位的CTL反應(yīng)明顯弱于L~d-限制性表位的CTL反應(yīng)。HSP70作為分子佐劑可以明顯改善多表位DNA疫苗的有效性。當(dāng)HSP70融合多表位抗原時，不僅HBsAg抗原特異性的CTL反應(yīng)被增強了，而且各個表位特異性(L~d、K~d和K~b)的CTL反應(yīng)也明顯的增強了。在HBV轉(zhuǎn)基因小鼠模型中，多表位DNA疫苗免疫可以明顯的清除血液中的HBsAg，并顯著下調(diào)了HBV DNA的水平。因此，，我們的研究表明基于T細胞表位的多表位DNA疫苗在治療慢性HBV感染方面是一個可以考慮的方案。 以往的研究表明，盡管HSP70可以改善DNA疫苗的有效性，但是由于HSP70是一個高度進化保守的分子，因此有引起自身免疫反應(yīng)的可能性。在這個研究中，我們選擇了兩個截短的HSP70分子——N′-端的HSP70_(1-360)和C′-端HSP70_(359-610)，研究它們對于HBV DNA疫苗引起的抗原特異性的免疫反應(yīng)的調(diào)節(jié)作用。實驗結(jié)果表明，只有HSP70_(359-610)融合的HBV DNA疫苗誘導(dǎo)了一個增強的HBsAg特異性的抗體反應(yīng)，同時，這個融合疫苗并沒有誘導(dǎo)HSP70的抗體反應(yīng)。有趣的是，HSP70_(359-610)不僅僅調(diào)節(jié)了HBsAg特異性的CTL反應(yīng)，還在H-2~d小鼠模型中，明顯增強了L~d-限制性表位的CTL反應(yīng)和D~d-限制性表位的CTL反應(yīng)。同時，HSP70_(359-610)融合DNA疫苗免疫調(diào)節(jié)Th反應(yīng)平衡偏向于Th1方向。在轉(zhuǎn)基因小鼠模型中，HSP70_(359-610)促進了HBV DNA免疫引起的HBsAg清除和HBV DNA下調(diào)。我們的研究結(jié)果表明，在HBV DNA疫苗設(shè)計中，截短的結(jié)核桿菌熱休克蛋白HSP70分子——HSP70_(359-610)，可以替代完整的HSP70分子作為分子佐劑。
[Abstract]:Although prophylactic subunit recombinant hepatitis B vaccine and some therapeutic drugs have been widely used in clinic, hepatitis B virus (HBV) infection is still a serious threat to human health. Because of the unique ability of inducing humoral and cellular immunity, DNA vaccine provides a new direction for the prevention and treatment of HBV infection. The results of phase I clinical trials in recent years have shown that HBV DNA vaccine can induce protective response in healthy population: in chronic HBV infected population, it can induce specific T cell response and eliminate HBV DNA. However, the HBV DNA vaccine used in these clinical trials encodes only a single antigen protein, S antigen or PreS2/S antigen. Moreover, the immunogenicity of DNA vaccine needs further improvement because of its unique antigen expression and presentation. Therefore, based on the T cell epitopes on HBV surface antigen, we designed a multiepitope DNA vaccine containing multiple cytotoxic T cell HBV epitopes and helper T cell epitopes, and studied its function. At the same time, the function of molecular adjuvant of heat shock protein HSP70 from Mycobacterium tuberculosis was studied. The results showed that the multiepitope HBV DNA vaccine induced a stronger HBsAg specific CTL reaction than the HBsAg DNA vaccine with single antigen gene. However, the CTL response of Dnd- restricted epitope was significantly weaker than that of Lnd- restricted epitope CTL reaction. HSP70 as a molecular adjuvant could significantly improve the effectiveness of multiepitope DNA vaccine. When HSP70 fused polyepitope antigen, not only the specific CTL reaction of HBsAg antigen was enhanced, but also the CTL reaction of each epitope specific, LndndKnd and Knb) was obviously enhanced. In HBV transgenic mice, the multiple epitope DNA vaccine immunization could significantly eliminate HBsAg in blood, and significantly down-regulate the level of HBV DNA. Therefore, our study shows that multiepitope DNA vaccine based on T cell epitopes is a potential alternative for the treatment of chronic HBV infection. Previous studies have shown that although HSP70 can improve the effectiveness of DNA vaccines, HSP70 is a highly evolutionarily conserved molecule, so it is possible to induce an autoimmune response. In this study, we selected two truncated HSP70 molecules, HSP701-360) and CS-terminal HSP70- (359-610), to study their regulatory effects on the antigen-specific immune response induced by HBV DNA vaccine. The results showed that only HSP70- (359-610) HBV DNA vaccine induced an enhanced HBsAg specific antibody response, and the fusion vaccine did not induce HSP70 antibody response. It is interesting to note that HSP70359-610) not only regulates the specific CTL response of HBsAg, but also enhances the CTL response of Lnd- restricted epitope and the CTL reaction of Dnd- restricted epitope in H-2d mouse model. At the same time, the balance of Th response in immunomodulation of DNA vaccine was inclined to the direction of Th1. In transgenic mice model, HSP70 tippon 359-610) promoted HBsAg clearance and HBV DNA down-regulation induced by HBV DNA immunization. Our results show that the truncated HSP70 molecule of the heat shock protein (HSP70) of Mycobacterium tuberculosis can replace the complete HSP70 molecule as a molecular adjuvant in the design of HBV DNA vaccine.
【學(xué)位授予單位】：中國科學(xué)技術(shù)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2006
【分類號】：R392

【共引文獻】

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5 吳傳勇;婁加陶;蔣廷旺;錢

本文編號：1980467