【作者】 刘勇；

【导师】 徐志凯；

【作者基本信息】 中国人民解放军第四军医大学 ， 微生物学， 2003， 博士

【摘要】 肾综合征出血热(HFRS)是一种由汉坦病毒属病毒引起，临床上主要表现为发热、出血和肾功能损害的急性病毒性传染病。我国是世界上HFRS疫情最为严重的国家，年发病人数为5-10万人，临床病情严重，病死率较高。目前尚无HFRS特异性治疗药物，预防性疫苗也还在试验中，HFRS的发病机理尚未最后阐明。 汉坦病毒有不同的型别，在我国流行的主要是汉滩型和汉城型。该病毒是一种有囊膜的单股负链RNA病毒，基因组分为大(L)、中(M)、小(S)3个节段，分别编码病毒的RNA依赖的RNA聚合酶、囊膜糖蛋白(G1和G2)以及核蛋白(NP)。研究表明，汉坦病毒的M基因编码的囊膜糖蛋白(GP)可刺激机体产生中和抗体，而对感染动物和人体起到保护作用；但GP免疫原性较弱，刺激产生的抗体出现晚，滴度不高。NP是该病毒结构蛋白中免疫原性最强的，其刺激机体产生的特异性抗体出现早、滴度高、维持时间长，并且还有诱导机体细胞 第四军医大学博士学位论文免疫应答的作用。 因此，汉坦病毒GP和NP在诱导机体免疫应答中可能均起重要作用，如何发挥汉坦病毒不同结构蛋白在诱导机体免疫应答中的优势互补作用，是HFRS基因工程疫苗研究中亟待解决的问题。 本研究在本室前期工作基础上，将汉滩病毒（HTNV）76一吕株编码 GI、GZ糖蛋白的M基因片段分别与编码NP氨基端aa 1247的S基因片段p．刀拼接，利用杆状病毒和腺病毒表达系统进行融合表达以及表达产物的免疫学分析。 l、成功构建了含有 HTNV GZSO．7嵌合基因片段的重组杆状病毒转移载体pFBHTa－GZSO＊，并用 CaCI。法转化 DH OBac，获得重组 Bacmid DNA，然后用LipofectAMINE jL转染sN昆虫细胞，获得重组杆状病毒Bac－GZSO．7并进行了初步鉴定。PCR、IFA、夹心ELISA及Western－blot证实重组杆状病毒成功感染sN细胞，并在细胞中有明显目的蛋白的表达。 2、将 PCR获得的含 HTNV GISO．7和 GZSO．7俄合基因片段分别克隆到腺病毒转移载体pshuttle中，然后通过特异性的ICeul和P！S 酶切后将重组转移载体与 Adeno－X载体病毒 DNA相连，电转化 E．colt JM 09并用特异 PCR进行了筛选和鉴定，获得重组腺病毒Adeno－GI SO．7和Adeno－GZSO．7的DNA，Pac！线性化后转染HEK293细胞得到重组腺病毒原种。进一步对重组腺病毒滴度和分别感染 HEK 293 和 Vero－E6 细胞的结果进行了 ELISA、IFA 和Western－blot初步分析，证实成功获得重组腺病毒，并且目的基因能在靶细胞中有效表达，具有良好的体外免疫学活性。 3、分别以重组Bac－GISO．7和 Bac－GZSO．7杆状病毒感染sfg细胞裂解液粗蛋白免疫小鼠，以重组 Adeno－GI SO．7 FI Adeno－G2507腺病毒腹腔注射免疫Balb／c小鼠，然后以IFA、ELISA、微量细胞培养中和试验及T淋巴细胞增殖试验检测免疫效果。结果显示：口）Bac－G旧0．7和 Bac－GZSO．7感染妞细胞裂解液免疫可刺激小鼠产叫二高滴度的 NP特异性结合抗体，效价可达到 1：3200以上，而针对＊卜的效价也可达到1：200，表明机体对GP和＊P片段均产生免疫 一4． 第四军医大学博士学位论文反应。两组融合蛋白还均可刺激机体产生低水平O：20）的中和抗体和明确的淋巴细胞增殖反应。p）Adeno－GI SO．7和 Adeno－GZSO．7重组腺病毒免疫均可刺激小鼠产生低滴度的抗NP口：160和 1：320）和抗GP抗体（：20和 1：40X 同时也可产生效价约1：5刁＊ 的低滴度中和抗体并刺激T淋巴细胞增殖反应。 本研究结果表明，无论是杆状病毒还是腺病毒表达系统均能在靶细胞或动物体内成功表达 HTNV 76－118株 GI SO．7和 GZSO．7嵌合基因；无论是融合蛋白免疫还是重组病毒直接免疫，II TNTNV 76－118株GI SO．7和GZSO．7 M合基因或融合蛋白均能有效刺激机体的体液免疫和细胞免疫应答。随着两种表达系统的不断发展和完善，随着靶基因的改造和修饰技术的不断进步，使用嵌合基因及其编码融合蛋白免疫的策略将有可能为发展为一种有效的预防HFRS的基因工程疫苗提供有用的理论依据和实验基础。更多还原

【Abstract】 Hemorrhagic Fever with Renal Syndrome (HFRS) is an acute infectious disease characterized by fever, hemorrhage and nephritis which is caused by Hantavirus. In China about 50,000 -100,000 people per year were infected by Hantavirus with the severe symptoms and high mortality. There are still no special therapeutical drugs and prophylactic vaccines directed to HFRS, and the etiologic mechanism of HFRS is not clearly elucidated until now.Hantavirus includes several serologically distinct viruses. Serologic and genetic study suggested that the most cases of HFRS in China were caused by hantaan (HTNV) or seoul (SEOV) serotype of Hantavirus. The Hantavirus is a single-stranded negative-sense RNA virus genus. The genome of the virus is composed of L, M and S segment which encoded RNA dependent RNA polymerase (RDRP), envelope glycoprotein G1 and G2, and nucleoprotein (NP) respectively. It is indicated that the glycoprotein (GP), which was encoded by M segment, couldstimulate organism to produce neutralization antibody and could protect infected animal and human body from HTNV lethal infection. But the immunogenicity of GP is weak. The antibody stimulated by GP is produced later and the liter is low. Whereas the NP, another structural protein, has the strongest immunogenicity and can stimulate an early, high-titer and long-lasting antibody response, and plays an important role in inducing cellular immunity activities against HTNV infection in vivo.Therefore GP and NP may play equal important role in stimulating the humoral or cellular immunity. How to bring each advantages into full play in inducing immune response in vivo is a problem waiting to be solved in HFRS genetic engineering vaccine.In this research, Gl, G2 fragment of M segment and 0.7Kb fragment of S segment 5" terminal of the HTNV 76-118 strain were spliced respectively, and then their fusion expression and immunological characterization were tested using baculovirus and adenovirus expression system respectively.1. The recombinant donor plasmid pFBHTa-G20.7 was constructed firstly. Then the recombinant Bacmid DNA and baculovirus Bac-G2S0.7 were harvested after transforming DHIOBac and transfecting sf9. PCR, IFA, HLISA and western-blot were used to identify the characterization of Bac-G2S0.7 in vitro. The results suggested that the fusion protein was successfully expressed in the sf9 cells.2. The PCR generated HTNV G1S0.7 and G2S0.7 chimeric gene were cloned to pShuttle vector, and connected with Adeno-X DNA after I-CeuI and Pl-Scel digestion, respectively. The recombinant Adeno-G1S0.7 and Adeno-G2S0.7 DNA were harvested by elctrotransformation of E.coli JM109 and identified by PCR. The original recombinant adenoviruses were then harvested by transfection of the package cells HEK. 293 using the Pad digested linear adenovius DNA. EL1SA, IFA and western-blot analysis results suggested that the recombinant adenovirus can infect HEK.293 and Vero-l? cells and express chimeric genes in the cells respectively, and the expressed fusion protein keep its biological activity in vitro.3. The freeze-thawing proteins of sf9 infected by recombinant Bac-GlS0.7 or Bac-G2S0.7 baculovirus, and the recombinant Adeno-GlS0.7 or Adeno-G2S0.7 adenovirus were used to immune the Balb/c mice, respectively. The immune response was tested by IFA, ELISA, microcell-culture neutralizing experiment and T lymphocyte proliferation test (MTT assay). The results showed: (1) the fusion protein of sf9 cells infected by Bac-GlS0.7 or Bac-G2S0.7 can stimulate high-title combining antibodies against NP (>1:3200) and GP (1:200) respectively detected by ELISA, which suggested both GP and NP immune response were activated. The low-titer neutralization antibody reaction (1:20) and clear lymphocyte proliferation were detected simultaneity. (2) the low-title combining antibodies against NP (1:160/1:320) and GP(1:20/1:40) were evoked by administration of recombinant Adeno-GlS0.7 or Adeno-G2S0.7 adenovirus respectively, and similar results of neutralization antibody activity (1:5-1:10) a更多还原