本文選題：SARS冠狀病毒 + 蛋白表達�。� 參考：《華南熱帶農(nóng)業(yè)大學》2005年碩士論文

【摘要】：嚴重急性呼吸道綜合癥(severe acute respiratory sydrome, SARS)的病原體為SARS冠狀病毒,SARS-CoV中參與病毒組裝和細胞侵染過程的物質(zhì)主要為4種結(jié)構(gòu)蛋白(S、M、N、E),這些蛋白是決定SARS-CoV獨特的生物學行為的物質(zhì)基礎(chǔ),正確表達這四種結(jié)構(gòu)蛋白對于SARS的診斷,試劑開發(fā)和疫苗的構(gòu)建有著深遠的意義。 本課題研究的目的是用大腸桿菌表達的SARS冠狀病毒的主要結(jié)構(gòu)蛋白S、M、N、E作為抗原檢測SARS-CoV抗體的可行性,及其這4個結(jié)構(gòu)蛋白基因構(gòu)建的DNA疫苗誘發(fā)小鼠產(chǎn)生抗SARS-CoV的IgG水平。研究方法:首先克隆了SARS-CoV主要結(jié)構(gòu)蛋白(S、M、N、E)的編碼基因,以pET-15b、pET-22b、pQE-2為載體,并分別在原核系統(tǒng)中表達,表達的蛋白均以可溶的形式存在,根據(jù)融合蛋白的性質(zhì)采用親和層析的方法獲得純化的重組蛋白M15b、M22b、rN、rS1、rS2、E15b。同時將這四個主要蛋白的編碼基因克隆入真核表達載體pSecTagB,構(gòu)建DNA疫苗,免疫小鼠后獲得抗血清,并進行了一系列的ELISA檢測。結(jié)果發(fā)現(xiàn)以重組蛋白作為抗原進行ELISA檢測能與免疫了DNA疫苗的小鼠血清以及SARS病人恢復期血清均可發(fā)生特異性的抗原抗體反應;另外,用SARS全病毒作為抗原(華大公司SARS檢測試劑盒)也可以和免疫鼠血清發(fā)生抗原抗體特異性反應。重組蛋白可以在大腸桿菌中得到高效表達,并可作為SARS檢測的抗原。構(gòu)建的DNA疫苗可以在小鼠體內(nèi)產(chǎn)生抗SARS的特異性抗體,為SARS-CoV DNA疫苗的開發(fā)提供借鑒作用。
[Abstract]:The pathogen of severe Acute Respiratory Syndrome (SARS) is SARS-CoV SARS-CoV, which is involved in the assembly and infection of SARS-CoV mainly by four structural proteins, which are the material basis for determining the unique biological behavior of SARS-CoV. The correct expression of these four structural proteins is of great significance for SARS diagnosis, reagent development and vaccine construction. The purpose of this study was to detect the SARS-CoV antibody by using the main structural protein of SARS coronavirus expressed in Escherichia coli as antigen, and the level of IgG against SARS-CoV in mice induced by the DNA vaccine constructed by these four structural protein genes. Methods: firstly, the encoding gene of SARS-CoV main structural protein (SARS-CoV) was cloned. The vector pET-15bPET-22bPQE-2 was expressed in prokaryotic system, and the expressed proteins were all in soluble form. According to the properties of the fusion protein, the purified recombinant protein M15bS1, rS1 and E15b was obtained by affinity chromatography. At the same time, the four major protein coding genes were cloned into eukaryotic expression vector pSecTagB. the DNA vaccine was constructed, and the antiserum was obtained after immunizing mice, and a series of Elisa tests were carried out. The results showed that Elisa with recombinant protein as antigen could react with the sera of mice immunized with DNA vaccine and the convalescent sera of SARS patients. SARS-CoV as antigen (SARS-CoV kit) can also react specifically with the sera of immunized mice. The recombinant protein can be highly expressed in E. coli and can be used as an antigen for SARS detection. The constructed DNA vaccine can produce specific antibodies against SARS in mice and provide reference for the development of SARS-CoV DNA vaccine.
【學位授予單位】：華南熱帶農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2005
【分類號】：R392

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