本文關(guān)鍵詞： A型流感病毒 核蛋白 重組質(zhì)粒 原核表達(dá)　出處：《鄭州大學(xué)》2005年碩士論文　論文類型：學(xué)位論文

【摘要】：背景與目的 A型流感病毒含有8條分離的負(fù)義RNA基因組片斷,核蛋白(nucleoprotein, NP)由節(jié)段5編碼。NP與病毒的RNA及多聚酶共同裝配成RNP復(fù)合體,通過(guò)NP與RNA、其它病毒蛋白成分及感染細(xì)胞上某些大分子之間的相互作用,影響病毒的轉(zhuǎn)錄、復(fù)制、裝配及轉(zhuǎn)運(yùn)功能,而病毒的復(fù)制與轉(zhuǎn)錄直接關(guān)系到對(duì)宿主的侵害,是病毒感染與致病的重要因素。另外核蛋白是細(xì)胞毒T淋巴細(xì)胞(Cytotoxic T lymphocytes, CTL)識(shí)別的主要抗原,CTL通過(guò)識(shí)別感染細(xì)胞表面的由MHC Ⅰ類分子提呈的病毒抗原肽,有利于清除病毒及控制感染。由于核蛋白基因相當(dāng)保守,其抗原肽誘導(dǎo)的細(xì)胞免疫反應(yīng)能夠針對(duì)同型不同亞型的流感病毒株產(chǎn)生交叉反應(yīng)。當(dāng)前的流感病毒亞單位疫苗主要利用表面抗原誘導(dǎo)體液免疫,抗體的株特異性及表面抗原的易變性使疫苗具有很大局限性。因此若能將核蛋白或其抗原肽作為疫苗成分來(lái)誘導(dǎo)產(chǎn)生CTL反應(yīng),將有可能提供更加廣泛、更加有效的保護(hù)作用。 本實(shí)驗(yàn)擬利用流感病毒株A/Swine/Henan/703/2001(H3N2)構(gòu)建原核重組質(zhì)粒pGEX-4T-1-NP,并誘導(dǎo)該原核重組質(zhì)粒在大腸桿菌中表達(dá)核蛋白,從而為進(jìn)一步探討核蛋白與宿主免疫反應(yīng)的關(guān)系、利用核蛋白構(gòu)建病毒亞單位疫苗及其它與核蛋白相關(guān)的病毒功能方面研究提供實(shí)驗(yàn)基礎(chǔ)。 材料與方法 本實(shí)驗(yàn)從流感病毒株A/Swine/Henan/703/2001(H3N2)中提取總RNA,通過(guò)RT-PCR擴(kuò)增到帶有EcoRI酶切位點(diǎn)的NP全長(zhǎng)基因序列;將NP基因片段與pGEM-T Easy克隆質(zhì)粒連接,將重組子轉(zhuǎn)化JM109感受態(tài)菌,利用Amp抗性、藍(lán)白篩選
[Abstract]:Background and purpose. Influenza A virus contains eight isolated negative sense RNA genomic fragments. The nucleoprotein nucleoprotein (NPN) is encoded by segment 5. NP is assembled into a RNP complex with the virus's RNA and polymerase. Through the interaction between NP and RNAs, other viral protein components and some macromolecules in infected cells, the transcription, replication, assembly and transport of viruses are affected, and the replication and transcription of viruses are directly related to the invasion of the host. Nuclear protein is the main antigen recognized by cytotoxic T lymphocytes (CTLs) by recognizing viral antigenic peptides presented by MHC class I molecules on the surface of infected cells. Help to clear the virus and control infection. Because the nucleoprotein gene is quite conserved, The cellular immune response induced by its antigenic peptide can cross-react with influenza virus strains of the same type and different subtypes. The current influenza virus subunit vaccine mainly uses surface antigen to induce humoral immunity. The specificity of antibody and the variability of surface antigen make the vaccine have great limitation, so if the nucleoprotein or its antigen peptide can be used as vaccine component to induce the CTL reaction, it will be possible to provide more extensive and effective protective effect. In this experiment, the prokaryotic recombinant plasmid pGEX-4T-1-NPN was constructed by using influenza virus strain A / Swine / Henan / 703 / 2001 H3N2, and the prokaryotic recombinant plasmid was induced to express the nucleoprotein in Escherichia coli, so as to further study the relationship between the nucleoprotein and host immune reaction. Construction of virus subunit vaccine using nucleoprotein and other viral function related to nucleoprotein provide experimental basis. Materials and methods. In this experiment, total RNAs were extracted from influenza virus strain A / Swine / Henan / 703 / 2001 H3N2, and the full-length NP gene sequence with EcoRI restriction site was amplified by RT-PCR. The NP gene fragment was ligated with pGEM-T Easy clone plasmid, the recombinant plasmid was transformed into JM109 receptive bacteria, Amp resistance was used and blue-white screening was carried out.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2005
【分類號(hào)】：R373

【共引文獻(xiàn)】

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