本文選題：家貓血管緊張素轉換酶2(fACE2) + SARS-CoV�。� 參考：《華中農業(yè)大學》2006年碩士論文

【摘要】：2002年冬天至2003年，我國爆發(fā)嚴重急性呼吸綜合癥，并迅速傳播至全球二十多個國家，導致8000多人感染，將近10％感染者死亡，一種新的冠狀病毒(SARS-CoV)是這場疫情的元兇。SARS-CoV的傳播與動物有關。2003年，研究人員從果子貍中分離到了SARS病毒，全基因組序列與人類SARS病毒具有99％的同源性。最近研究又發(fā)現(xiàn)蝙蝠攜帶有類SARS-CoV，與人SARS-CoV基因組序列同源性達92％，不過這種病毒不會直接感染人類。野生蝙蝠可能是非典病毒的源頭宿主，果子貍只不過是SARS-CoV變異傳播鏈上一個很重要的環(huán)節(jié)，作為SARS-CoV的中間宿主和重要載體將病毒從野外傳染到人類身上。家貓也是SARS-CoV的易感動物。2003年SARS大爆發(fā)期間，在香港陶大花園SARS患者家中的寵物貓血清中檢測到了SARS-CoV。SARS-CoV人工感染家貓、雪貂也獲得成功，病毒能夠在家貓體內正常增殖，被感染的家貓能有規(guī)律排毒，出現(xiàn)與人類SARS-CoV感染類似的病理變化，并可將病毒傳染給與其共同飼養(yǎng)的原本不帶毒的動物。 人的血管緊張素轉換酶2(ACE2)是SARS-CoV的一個功能性受體，本實驗室首次克隆了家貓ACE2基因，，為了深入探討家貓ACE2基因作為SARS-CoV受體及其在SARS傳播中的作用，本研究開展了如下工作： (1) 家貓ACE2空間結構建模：家貓ACE2基因編碼的氨基酸序列與人ACE2同源性達85％，本研究以人ACE2結構為模板，用同源模建法建立了貓ACE2的三維空間結構模型，蛋白質對接法模擬了貓ACE2分子與SARS-CoVS的RBD結合。所預測的三維結構基本在一定程度上能反映出貓ACE2真實的空間構象，預測得到的模型與人ACE2的空間結構非常相似。比較ACE2分子中與SARS-CoV S蛋白受體結合區(qū)相接觸的18個氨基酸，家貓ACE2只有三個氨基酸與人ACE2不同，分別為L24Q、E38D與T82M，提示家貓ACE2可能為SARS-CoV的功能性受體。 (2) 家貓ACE2原核表達及與SARS-CoV S1蛋白的結合：人ACE2的受體功能區(qū)位于N端，SARS-CoV的受體結合蛋白為其纖突蛋白S1蛋白。為了獲得家貓ACE2作為SARS-CoV受體的直接證據(jù)，本研究在大腸桿菌BL21(DE3)中表達了家貓ACE2 N端19aa-367aa，并純化得到重組蛋白。經配體轉印試驗(ligand blotting assay)和ELISA均證實，重組的fACE2_(19-367)蛋白片段能夠與重組的SARS-CoV S1蛋白結合。 (3) 貓ACE2基因的真核表達：為了深入研究貓ACE2基因的受體活性，進一步構建了基因片段的真核表達載體pcDNA-mfA367，間接免疫熒光檢測表明家貓ACE2 N端laa-367aa在細胞膜上實現(xiàn)了定位表達。這為深入研究貓ACE2的SARS-CoV受體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.

【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2006
【分類號】：R346;Q78

【參考文獻】

相關期刊論文 前1條

1 費小戰(zhàn);盧海松;郭紅燕;譚亞娣;陳煥春;郭愛珍;;SARS冠狀病毒刺突蛋白S144-643的表達及免疫原性分析[J];華中農業(yè)大學學報;2006年01期

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