本文關(guān)鍵詞：結(jié)核桿菌hsp70在乙腦基因工程疫苗研制中的免疫增強(qiáng)作用　出處：《南京農(nóng)業(yè)大學(xué)》2006年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 結(jié)核桿菌熱休克蛋白70 乙型腦炎 E蛋白主要抗原片段 中和表位 Th1反應(yīng)

【摘要】：流行性乙型腦炎(簡(jiǎn)稱乙腦)屬于黃病毒科，引起人類或馬的中樞神經(jīng)系統(tǒng)感染以及豬的流產(chǎn)，能通過(guò)持續(xù)感染的蚊子傳播給人，并且主要在脊椎動(dòng)物宿主內(nèi)存在，特別是具有較高病毒血癥的豬和鳥類，在人類乙腦主要引起致死率較高的腦炎，致死率從20-50％，但是絕大多數(shù)的幸存者都具有中樞神經(jīng)系統(tǒng)的后遺癥；在家養(yǎng)動(dòng)物如豬和馬，主要引起腦炎，流產(chǎn)甚至死亡。與黃病毒科的其它成員類似，為單鏈正股RNA，長(zhǎng)度為11kb，編碼一個(gè)含多聚蛋白的開(kāi)放性閱讀框，這個(gè)多聚蛋白可通過(guò)蛋白酶切割成至少11個(gè)蛋白，該病毒的結(jié)構(gòu)蛋白包括衣殼蛋白，膜蛋白和囊膜蛋白，位于5’端三分之一的區(qū)域，而非結(jié)構(gòu)蛋白從NS1到NS5位于閱讀框其余的區(qū)域。 目前用于豬的乙腦疫苗包括弱毒苗和滅活苗，但是有關(guān)它們的安全性，生產(chǎn)和使用費(fèi)用等問(wèn)題越來(lái)越突出。WHO正在呼吁改善和研制新型乙腦疫苗，乙腦病毒的E蛋白與病毒和細(xì)胞受體結(jié)合，膜融合有關(guān)，也是引起中和抗體及宿主保護(hù)的主要免疫蛋白。結(jié)核桿菌熱休克蛋白70具有分子佐劑和載體效應(yīng)，是結(jié)核桿菌的主要抗原成分之一，可誘導(dǎo)和增強(qiáng)機(jī)體體液免疫和細(xì)胞免疫的發(fā)生，，因此用該基因與一些傳染病或寄生蟲病病原體的抗原基因融合表達(dá)以制備疫苗，借助它的載體分子效應(yīng)和佐劑作用，以提高病原體抗原的免疫原性和疫苗的免疫預(yù)防效果。 本文將結(jié)核桿菌熱休克蛋白70與乙型腦炎E蛋白主要抗原片段在畢赤酵母中進(jìn)行融合表達(dá)免疫小鼠，用以研究hsp70對(duì)免疫反應(yīng)的影響，此外還對(duì)結(jié)核桿菌hsp70與抗原肽以何種方式共同作用才能發(fā)揮其佐劑效應(yīng)進(jìn)行了研究，根據(jù)報(bào)道結(jié)核桿菌hsp70分為兩個(gè)區(qū)域，N末端為ATPase區(qū)，C末端為肽連接區(qū)，其中肽連接區(qū)為hsp70中真正行使佐劑功能的區(qū)域，因此本文將E蛋白主要抗原片段與肽連接區(qū)進(jìn)行融合表達(dá)并免疫，研究肽連接區(qū)的分子佐劑功能；另外，將已報(bào)道的E蛋白上的中和表位與結(jié)核桿菌hsp70在大腸桿菌中融合表達(dá)，將其免疫效果與乙腦弱毒苗SA14-14-2進(jìn)行比較。實(shí)驗(yàn)結(jié)果如下： 1，結(jié)核桿菌熱休克蛋白70及其功能域肽連接區(qū)對(duì)乙型腦炎E蛋白主要抗原片段引起的免疫反應(yīng)的影響。 在本實(shí)驗(yàn)室已構(gòu)建的原核表達(dá)載體(含乙腦疫苗株SA14-14-2株E蛋白基因主要抗原片段)的基礎(chǔ)上用巴斯德畢赤酵母系統(tǒng)表達(dá)，該片段長(zhǎng)1113bp，編碼371個(gè)氨基
[Abstract]:Japanese encephalitis (JE) belongs to the family Flaviviridae, causing infections of the central nervous system and the nerve of human or pig abortion horse, through the persistent infection of mosquitoes to humans, and mainly in the vertebrate host in memory, especially with high viremia in pigs and birds in the human Japanese encephalitis mainly caused a high mortality rate. Encephalitis, the death rate from 20-50%, but most of the survivors have central nervous system sequelae; in the domestic animal such as pigs and horses, mainly caused by encephalitis, abortion and even death. With other members of the Flaviviridae family, single stranded positive strands of RNA, length of 11KB, encoding a polyprotein open reading frame of the multimeric protein can be cut into at least 11 protein by protease, structural proteins of the virus including capsid protein, membrane protein and membrane protein, is located in the 5 'end of 1/3 areas, Non structural proteins, from NS1 to NS5, are located in the rest of the reading frame.
At present for swine JE vaccine including attenuated vaccine and inactivated vaccine, but their safety, production costs and the use of more and more prominent issues such as.WHO are calling for improvement and development of a new type of JE vaccine, Japanese encephalitis virus E binding protein and virus and cellular receptors, membrane fusion, is mainly caused by the immune protein neutralizing antibody and the host protection. Mycobacterium tuberculosis heat shock protein 70 with molecular adjuvant and carrier effect, is one of the major antigens of Mycobacterium tuberculosis, which can induce and enhance the humoral and cellular immunity, so the antigen gene gene and some infectious or parasitic disease pathogen fusion protein to prepare a vaccine, with the help of it the carrier molecule effect and immune adjuvant effect, to improve the primary pathogen antigen and vaccine prevention effect.
This will be the main antigen fragment of Mycobacterium tuberculosis heat shock protein 70 and E protein of Japanese encephalitis in mice immunized with fusion expression in Pichia pastoris, and used to study the effects of HSP70 on immune response, in addition to the Mycobacterium tuberculosis HSP70 antigen peptide and the manner in which the interaction can play its adjuvant effect was studied, according to the reports of Mycobacterium tuberculosis Hsp70 is divided into two regions, N terminal ATPase, C terminal peptide linker region where peptide connection region for regional real exercise adjuvant function in HSP70, so the antigen peptide E protein fragment and the connection region for the fusion expression and immune function, molecular adjuvant peptide of the connecting region; in addition, the E protein has been reported on the neutralizing epitope of Mycobacterium tuberculosis and HSP70 fusion expression in Escherichia coli, the immune effect and Japanese encephalitis attenuated vaccine SA14-14-2 were compared. The experimental results are as follows:
1, the effect of the heat shock protein 70 and its functional domain peptide junction on the immune response caused by the main antigen fragment of E protein in Japanese encephalitis.
On the basis of the prokaryotic expression vector (containing the main antigen fragment of E gene of SA14-14-2 strain of Japanese encephalitis vaccine strain), it was expressed in Pichia pastoris system. The fragment is long 1113bp and encodes 371 amino groups, which is constructed in our laboratory.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)期刊論文 前1條

1 王軼男;賈立軍;薛書江;胡詩(shī)悅;錢年超;張守發(fā);;牛瑟氏泰勒蟲熱休克蛋白70的克隆與原核表達(dá)重組質(zhì)粒的構(gòu)建[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2013年02期

相關(guān)碩士學(xué)位論文 前1條

1 孔Z腪

本文編號(hào)：1376092