家猫血管紧张素转换酶2基因表达、突变体构建及作为SARS-CoV受体的功能研究

发布时间：2018-05-10 06:43

本文选题：家猫血管紧张素转换酶2(fACE2) + SARS-CoV　；参考：《华中农业大学》2006年硕士论文

【摘要】：2002年冬天至2003年，我国爆发严重急性呼吸综合症，并迅速传播至全球二十多个国家，导致8000多人感染，将近10％感染者死亡，一种新的冠状病毒(SARS-CoV)是这场疫情的元凶。SARS-CoV的传播与动物有关。2003年，研究人员从果子狸中分离到了SARS病毒，全基因组序列与人类SARS病毒具有99％的同源性。最近研究又发现蝙蝠携带有类SARS-CoV，与人SARS-CoV基因组序列同源性达92％，不过这种病毒不会直接感染人类。野生蝙蝠可能是非典病毒的源头宿主，果子狸只不过是SARS-CoV变异传播链上一个很重要的环节，作为SARS-CoV的中间宿主和重要载体将病毒从野外传染到人类身上。家猫也是SARS-CoV的易感动物。2003年SARS大爆发期间，在香港陶大花园SARS患者家中的宠物猫血清中检测到了SARS-CoV。SARS-CoV人工感染家猫、雪貂也获得成功，病毒能够在家猫体内正常增殖，被感染的家猫能有规律排毒，出现与人类SARS-CoV感染类似的病理变化，并可将病毒传染给与其共同饲养的原本不带毒的动物。人的血管紧张素转换酶2(ACE2)是SARS-CoV的一个功能性受体，本实验室首次克隆了家猫ACE2基因，，为了深入探讨家猫ACE2基因作为SARS-CoV受体及其在SARS传播中的作用，本研究开展了如下工作： (1) 家猫ACE2空间结构建模：家猫ACE2基因编码的氨基酸序列与人ACE2同源性达85％，本研究以人ACE2结构为模板，用同源模建法建立了猫ACE2的三维空间结构模型，蛋白质对接法模拟了猫ACE2分子与SARS-CoVS的RBD结合。所预测的三维结构基本在一定程度上能反映出猫ACE2真实的空间构象，预测得到的模型与人ACE2的空间结构非常相似。比较ACE2分子中与SARS-CoV S蛋白受体结合区相接触的18个氨基酸，家猫ACE2只有三个氨基酸与人ACE2不同，分别为L24Q、E38D与T82M，提示家猫ACE2可能为SARS-CoV的功能性受体。 (2) 家猫ACE2原核表达及与SARS-CoV S1蛋白的结合：人ACE2的受体功能区位于N端，SARS-CoV的受体结合蛋白为其纤突蛋白S1蛋白。为了获得家猫ACE2作为SARS-CoV受体的直接证据，本研究在大肠杆菌BL21(DE3)中表达了家猫ACE2 N端19aa-367aa，并纯化得到重组蛋白。经配体转印试验(ligand blotting assay)和ELISA均证实，重组的fACE2_(19-367)蛋白片段能够与重组的SARS-CoV S1蛋白结合。 (3) 猫ACE2基因的真核表达：为了深入研究猫ACE2基因的受体活性，进一步构建了基因片段的真核表达载体pcDNA-mfA367，间接免疫荧光检测表明家猫ACE2 N端laa-367aa在细胞膜上实现了定位表达。这为深入研究猫ACE2的SARS-CoV受体功能提供了重要工具。
[Abstract]:From 2002 to 2003, severe acute respiratory syndrome broke out in China and spread rapidly to more than 20 countries around the world, resulting in more than 8000 people and nearly 10% infected people. A new coronavirus (SARS-CoV) was the.SARS-CoV of the outbreak of the epidemic and animal related.2003, and the researchers separated SAR from the beaver. S virus, the whole genome sequence is 99% homologous to the human SARS virus. Recent studies have found that bats carry the SARS-CoV like SARS-CoV, and the homology of the human SARS-CoV genome is 92%, but the virus does not directly infect humans. The wild bat may be the source of the SARS virus, and the beaver is only the SARS-CoV variant transmission A very important link in the chain, as the intermediate host and important carrier of SARS-CoV, infects the virus from the wild to the human body. Domestic cats are also the SARS-CoV susceptible animal.2003 SARS outbreak. In the pet cat serum of the SARS patients in the Hongkong pottery garden, the cat was detected by SARS-CoV.SARS-CoV artificially and the ferret was also obtained. Successfully, the virus can proliferate normally in the domestic cat, the infected domestic cat can have the regular detoxification, the pathological changes similar to the human SARS-CoV infection, and the virus can be transmitted to the original non poisonous animals.
Human angiotensin converting enzyme 2 (ACE2) is a functional receptor for SARS-CoV. The domestic cat ACE2 gene was first cloned in our laboratory. In order to explore the role of the domestic cat ACE2 gene as a SARS-CoV receptor and its role in the transmission of SARS, the following work has been carried out.
(1) modeling of ACE2 spatial structure of domestic cats: the amino acid sequences encoded by ACE2 gene in domestic cats and human ACE2 are 85%. The three-dimensional spatial structure model of cat ACE2 is established by homologous ACE2 structure. The protein docking method simulates the RBD binding of cat ACE2 molecules to SARS-CoVS, and the predicted three-dimensional structure is basically at the same time. To a certain extent, it can reflect the real spatial conformation of the cat ACE2. The predicted model is very similar to the spatial structure of human ACE2. Comparing the 18 amino acids in the ACE2 molecule with the SARS-CoV S protein receptor binding area, the domestic cat ACE2 has only three amino acids different from the human ACE2, which are L24Q, E38D and T82M respectively, suggesting that the cat ACE2 may be SARS-. Functional receptors of CoV.
(2) ACE2 prokaryotic expression and binding with SARS-CoV S1 protein in domestic cats: the receptor functional area of human ACE2 is located at the N terminal and the receptor binding protein of SARS-CoV is its fibrinolytic protein S1 protein. In order to obtain the direct evidence of the domestic cat ACE2 as a SARS-CoV receptor, the present study expressed the family cat ACE2 BL21 (DE3) in BL21 (DE3) and purified it. The recombinant protein. It was confirmed by ligand blotting assay and ELISA that the recombinant fACE2_ (19-367) protein fragment could be combined with the recombinant SARS-CoV S1 protein.
(3) eukaryotic expression of the cat ACE2 gene: in order to study the receptor activity of the cat ACE2 gene, the eukaryotic expression vector pcDNA-mfA367 of the gene fragment was further constructed, and the indirect immunofluorescence detection showed that the ACE2 N terminal laa-367aa in the domestic cat was located on the cell membrane. This provides a deep Study on the SARS-CoV receptor function of cat ACE2. Want a tool.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R346;Q78

【参考文献】