【作者】 胡贵方；

【导师】 俞守义； 吴小兵；

【作者基本信息】 中国人民解放军第一军医大学 ， 病原生物学， 2003， 博士

【摘要】 乙型肝炎是一种由乙型肝炎病毒(hepatitis B virus，HBV)感染引起的严重威胁人类健康的传染性疾病，目前全球范围内大约有超过3.5亿人受到HBV危害。大约有15～40％的HBV感染者最终会发展为肝细胞癌(hepatocellular carcinoma，HCC)、肝硬化和肝衰竭。我国是乙型肝炎主要的流行区，现患慢性乙型肝炎的病人已突破1000万。迄今为止，慢性乙型肝炎的治疗仍无良策，因此，研制新型HBV疫苗和寻求有效的治疗乙型肝炎的手段迫在眉睫。2型重组腺相关病毒(adeno-associated vires type 2，rAAV-2)作为一种大有希望的基因转移载体，因能有效转导多种组织和细胞而引起了人们的高度重视。为此，本研究以研制新型HBV疫苗为目的，构建了表达HBV外膜主蛋白、大蛋白基因的rAAV-2，初步研究了其免疫原性，并探讨了表达HBV抗原基因的rAAV-2用于以树突状细胞(dendritic cell，DC)为基础的慢性乙型肝炎免疫治疗的可行性。 采用PCR法从PTHBV—1质粒中扩增HBV(ayw亚型)外膜主蛋白(HBsAg)、大蛋白(LHBsAg)基因；将PCR扩增产物插入rAAV-2表达质粒pSNAV中，构建重组质粒pSNAV-HBsAg和pSNAV-LHBsAg；用脂质体转染的方法将重组质粒转入BHK—21细胞中，G418筛选得到转入重组质粒并能表达目的基因细胞系BHK-HBsAg和BHK-LHBsAg；用具有rAAV包装功能的重组单纯疱疹病毒(HSV1-rc/ΔUL2)感染BHK-HBsAg和BHK-LHBsAg，纯化后得到rAAV-2-HBsAg和rAAV-2-LHBsAg；用高效液相色谱仪(HPLC)检测和SDS—聚丙稀酰胺凝胶电泳(SDS-PAGE)检测了rAAV-2-HBsAg和rAAV-2-LHBsAg的纯度；以点杂交方法检测重组病毒的物理滴度；ELISA检测混合细胞系博士论文:，、HB、，卜膜主蛋。、、蛋。、重组腺，关病毒的构减巍免。性研究BHK一HBsAg和BHK~LHBsAg中表面抗原(HBsAg)的表达，rAA认HBsAg和rA戌认LHBsAg在BHK一21细胞和293细胞中的表达。结果表明，H卫LC分析显示主峰为总面积大于99%;SDS一PAGE电泳结果显示3条特征性条带，其间的杂蛋白量非常少;点杂交检测rA戌瞬2一HBsAg和rAA认2.LHBsAg的物理滴度分别为sxlo，‘和ZxlolZ virusp耐cles/nil(vp翔吐);混合细胞株BHK一HBsAg中HBsAg的表达量为28.6士6.7ng乃x ro6eells，BHK~LHBsAg中HBsAg的表达量为15.4士5.5 ng zsxl06eeus;rA戌认2~HBs^g和rAAv-2~LHBsAg感染BHK一21细胞和293细胞后均能检测到HBsAg的表达，表达量随感染复数(伽ltiplicityofinfection，MOI)的增加而升高。提示我们已经获得了高滴度、高纯度、在体外能有效地感染培养细胞的rAAV-2一HBsAg和rAAv-2.LHBSAg，为下一步工作奠定了基础。 本文首次报道了rA戌认2载体介导HBV抗原基因用于HBV疫苗的研究。通过肌肉注射、尾静脉注射、腹腔注射和胃饲给药途径，我们观察了rA入v-2~HBsAg和rAA认2.LHBsAg在小鼠体内的转导效率和诱导小鼠产生HBv抗原特异性的体液和细胞免疫反应的能力。结果显示，rAAv-2~HBsAg和rAAv-2.LHBsAg通过肌肉注射、尾静脉注射、腹腔注射和胃饲途径进入小鼠体内后能有效表达HBsAg，同一给药途径，rA戌瞬2.HBsAg和rA入叭2~LHBsAg在小鼠体内HBsAg的表达水平无明显差异(p>0 .05);同一重组病毒(rA戌认2一HBsAg或rAAV一2一LHBsAg)不同给药途径之间HBsAg的表达水平亦无明显差异(p>0 .05)。重组病毒通过不同途径在小鼠体内表达的同时，可在较长时间(80天)诱导小鼠产生HBV抗原特异性的体液和细胞免疫反应:不同给药途径之间的免疫效果比较发现，肌肉注射、尾静脉注射、腹腔注射和胃饲都能诱发机体产生HBV保护性抗体和激发CTL产生，但以尾静脉注射效果最好，腹腔注射、肌肉注射和胃饲三者之间差别不大 (p>0 .05);在各种给药途径中，免疫反应能否出现、以及免疫反应的强度与剂量(给予的重组病毒颗粒数)有关。结果提示，基于rAAV载体的HBV疫苗，对于防止HBV感染，尤其是作为慢性乙型肝炎的治疗性疫苗和应用于对现有HBV疫苗无反应的患者无疑具有潜在的应用价值，值得做进一步的系统研究。 近年来，以DC为基础的免疫治疗已成为抗肿瘤和抗感染等研究的热点，本文也探讨了表达HBV抗原基因的rAA认2载体用于DC为基础的慢性乙型肝炎免疫治疗的可行性。首先，我们用表达报告基因一绿色荧光蛋白(GFP)基因和荧光素酶(luciferase，Luc)的rA月认2(分别简称为rAA认GFP和rAAV-Luc)感染了人外周血单核细胞来源DC以观测感染效能，在激光共聚焦显微镜观察了F仃C博士论文:表达HBV外膜主蛋白、大蛋白的重组腺相关病毒的构建及其免疫原性研究(fluorescein 150而Ocyanate，FITC，异硫氰酸荧光素)标记的rAA认2进入Dc的动态过程;结果显示，激光共聚焦显微镜下观察到了rAAV-2能进入DC;只有当Mol(multiplicity of infeetion，感染复数)大于1 X losvp/c ell时，乙AAV-2一lue和rA戌认2一GFP才能有效感染DC，需较高的MOI值的重组病毒才能有效感染Dc，报告基因的表达水平在MOI值为1 X 105一5 x 106vPlcell之间呈剂量依赖趋势，此后再提高MOI值并不能明显提高表达水平，感染后的DC表达水平不高。表明rAA认2可以有效感染DC，但转导效率?更多还原

【Abstract】 Hepatitis B, caused by hepatitis B virus (HBV) infection, is an infectious disease that has been threatening public health and has affected more than 350 million people worldwide. Among which, hepatocirrhosis, liver failure, or hepatocellular carcinoma will be developed eventually in about 15 to 40 percent. HBV is heavily endemic in China as there are approximately 10 million chronically infected patients. It is in urgent need of excogitating new HBV vaccines and searching for effective therapeutic methods since there is no panacea for chronic HBV infection to date.Due to its non-pathogenicity, the ability to infect various types of cells and the capability to mediate long-term gene expression, recombinant adeno-associated virus type 2 (rAAV-2) has attracted tremendous interest as a promising vector for gene delivery. In this study we have developed an HBV vaccine and then studied its immunogenicity by using rAAV-2 vector expressing HBV envelope major protein and large protein. Meanwhile, we have probed into the feasibility that rAAV-2 was introduced to dendritic cell (DC)-based immunotherapy of chronic hepatitis B.Hepatitis B virus (subtype ayw) envelope major protein (HBsAg) and lager protein (LHBsAg) gene were amplified from PTHBV-1 by PCR and then cloned into the adeno-associated virus vector pSNAV, the recombinant pSNAV-HBsAg and pSNAV- LHBsAg were transfected into BHK-21 cell by using Lipofectamine?2000. Both mixed cell lines, BHK-HBsAg and BHK-LHBsAg which expressing HbsAg, were isolated by using G418 selection and were then infected with recombinant herpes simplex virus (HSVl-rc/AUL2) which can package the rAAV-2. rAAV-2-HBsAg and rAAV-2-LHBsAg were obtained after purification. HPLC and SDS-PAGE were used to monitor the purity of rAAV-2-HBsAg and rAAV-2-LHBsAg,respectively. The blot hybridization was used to determine the physical titers of rAAV-2-HBsAg and rAAV-2-LHBsAg. The results of SDS-PAGE and HPLC showed that the purities of the final rAAV-2 products were higher than 99%. The physical titers of rAAV-2-HBsAg and rAAV-2-LHBsAg were 5 ×1011and 2 × 1012 virus particles/ml(vp/ml), respectively. The expression level of HBsAg in BHK-HbsAg and BHK-LHBsAg, detected by ELISA(enzyme-linked immunosorbent assay), were 28.6 ±6.7 ng/5× 106cells and 15.4±5.8 ng/5×106cells. The expression of HBsAg in BHK-21 cells and 293 cells infected with rAAV-2-HBsAg and rAAV-2-LHBsAg can be detected, and the amounts of HBsAg expression elevated following the increasing of MOI (multiplicity of infection, MOI). These results indicate that rAAV-2-HBsAg and rAAV-2-LHBsAg can transduce various types of cultured cells efficiently in vitro.The transduction efficiencies of rAAV-2-HBsAg and rAAV-2-LHBsAg in vivo and their immunogenicities were studied via intramuscular injection (ini), intravenous injection (iv), intraperitoneal injection (ip) and gastric gavage (gg). The results showed that there is no difference of HBsAg expression between rAAV-2-HBsAg and rAAV-2-LHBsAg via the same administration route. Expression levels of HBsAg are about the same after the administration of rAAV-2-HBsAg or rAAV-2-LHBsAg to Balb/c mice via different routes. One single administration (im, iv, ip and gg) of rAAV-2-HBsAg or rAAV-2-LHBsAg can induce both humoral and cellular immune response for a long time. Among the four administration routes, both rAAV-2-HBsAg and rAAV-2-LHBsAg can induce the strongest cytotoxic T lymphocyte (CTL) response via intravenous injection. To our knowledge, this is the first report that rAAV-2-mediated HBV antigen gene expression can induce CTL responses as well as anti-HBsAg antibodies. Thus, the rAAV-2 vector can be used as a promising candidate for hepatitis B vaccine, particularly as a therapeutic vaccine for chronic hepatitis B and for those patients who have non-responsive to the current HBV vaccine.Much attention has been paid to the adjuvanticity of DC in generating antigen-specific immune responses for anti-tumor and anti-infection therapeutics. First, Viral vectors have been introduced succes更多还原