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树鼩免疫球蛋白G的分离纯化及其兔抗血清的制备以及树鼩CCR55'端的克隆和序列分析
韦彦余
学位类型硕士
导师赵民安
2004-06-10
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业微生物学
关键词树韵 Hiv-1受体 Hiv-1辅助受体 序列分析 Fplc系统
摘要对于丙型肝炎病毒(HCV)和艾滋病病毒(HIV-1)的研究,函待解决的问题 之一是建立一种经济易得、对HCV/HIV-1易感并且发病症状与人类相似的动物 模型,以便对HCV/HIV-1的发病机理及药物筛选和疫苗进行深入研究。树晌(Tupaia,Tree shrew)是一种大量分布于云南、广西一带,易饲养,繁殖快, 分类学上接近于灵长目的小动物,已广泛用于生物医学方面的研究。有证据表明树 韵可以感染HCV。树韵细胞可以支持HIV-1进入靶细胞后复制循环的其它步骤, 但是我们实验室发现树晌细胞并不能感染Hlv-1,推测树晌的受体CD4或辅助受 体ccRS和cxcR4与人的相应的受体可能存在较大差异。目前我们实验室已经 得到了树韵的CXcR4,序列分析发现,树晌CXCR4与人的CXCR4差别不大, 可能不是限制HIV-1进入树韵免疫细胞的原因。为此,我们根据实验室以前得到 的树勃CCRS基因的中间一段,采用5'RACE技术克隆得到了CCR55’端。此 段含有决定树晌CCRS能否支持HIV-1进人宿主细胞的关键区域,由cDNA推断 出的氨基酸片段,发现在决定树韵ccRS能否支持HIV-1进人宿主细胞的关键位 点:Tyr-3,-10,-14,-15上与人ccRS差别较大,这可能是树韵不易感HIV-1的原因 之一。作为一种新型的实验动物模型,树韵的许多生理生化、遗传背景等研究还不 够充分。树韵的免疫球蛋白的分离纯化和抗体制备对建立动物模型是必需的。在 本实验中,我们采用即Lc系统结合自装柱的方法,分离纯化得到了高纯度的树 韵免疫球蛋白G,并尝试了制备抗树前免疫球蛋白G的抗体,但是由于对树韵免 疫球蛋白G特性不了解,免疫动物的结果不是很成功,得到的抗体的滴度较低。
其他摘要One of the imperative tasks in HCV/HTV-1 researches is to built an economical, easily infected animal model with human symptoms for investigating pathogenesis of disease and many vaccine strategies. Tree shrews (Tupaia belangeri), a small animal closely related to primates, are endemic to areas of Yunnan and Guangxi. The animal is easily bred, quickly reproduced and is widely used in biomedical researches due to susceptible to many medically important viruses. Now,research has been done that tree shrew could be infected by HCV. Research has been done which proved that tree shrews apparently supported postentry events in HIV-l infection.Our laboratory research proved tree shrew irnmunocytes couldn't be infected by HIV-l in vitro and deduced the structure differences of the HIV-l receptor CD4 and coreceptor CCR5 or CXCR4 between human and tree shrew irnmunocytes.Now our laboratory has gotten CXCR4 gene of tree shrew .After sequence analyses,we found out that CXCR4 of both tree shrew and human has no obvious difference.So tree shrew CXCR4 is likely to be a reason of restricting HIV-l entering tree shrew immunocytes.So tree shrew CCR5 5'end was analysed by the technique of 5'rapid amplification of cDNA ends.Leaders were designed according to a middle fragment in cDNA of tree shrew CCR5 .which we have gotten before.The fragment have gotten in the 5'end might be responsible to HIV-l infection.Its amino acid sequence has- been deduced according to cloned CCR5 cDNA.we found that key animo acids:Tyr-3,-10,-14r15, might be one of the reason for tree shrew's resistance to HIV-l infection,because there are have great differences between human and tree shrew CCR5. As a new animal model,the reaserch about physiology, heredity of tree shrew has not been carried out thoroughly.Purification of immunoglobuiin G in tree shrew and preparation rabbit-anti-tree-shrew-IgG serum are useful. In our experiment,we purified tree shrew IgG using FPLC system with chromatography column filled with general matrix and tried to prepare anti-tree-shrew-IgG serum.But it is not very successful to immunize the animal using tree shrew IgG as antigene,for we were unconversant about the character of tree shrew IgG.The quantity of antibodies we have gotten is very few.
文献类型学位论文
条目标识符http://ir.xjipc.cas.cn/handle/365002/4454
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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韦彦余. 树鼩免疫球蛋白G的分离纯化及其兔抗血清的制备以及树鼩CCR55'端的克隆和序列分析[D]. 北京. 中国科学院研究生院,2004.
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