【摘要】：破傷風(fēng)是由破傷風(fēng)毒素引起的一種嚴(yán)重的痙攣性疾病,有效的預(yù)防方法是接種破傷風(fēng)類毒素疫苗。傳統(tǒng)的類毒素疫苗是破傷風(fēng)毒素經(jīng)甲醛脫毒、精制而成的,現(xiàn)仍存在一些問題:接種類毒素后有一定的副反應(yīng)發(fā)生;破傷風(fēng)梭菌可形成芽孢形式,毒性高,生產(chǎn)疫苗有一定危險性;甲醛處理類毒素容易造成污染;化學(xué)處理后的類毒素可能發(fā)生毒性逆轉(zhuǎn)。因此,現(xiàn)有的類毒素疫苗還需進(jìn)一步改進(jìn)和發(fā)展,開發(fā)新型的基因工程疫苗是方向之一。本研究以破傷風(fēng)毒素C片段基因?yàn)榛A(chǔ),用大腸桿菌表達(dá)系統(tǒng)得到了高效表達(dá)的融合蛋白,之后又用沙門氏菌表達(dá)系統(tǒng)構(gòu)建了運(yùn)送破傷風(fēng)毒素C片段的重組減毒鼠傷寒沙門氏菌菌株,為研制新型的破傷風(fēng)疫苗奠定了基礎(chǔ)。 1.破傷風(fēng)毒素C片段基因的克隆及在大腸桿菌中的表達(dá) 本研究對破傷風(fēng)毒素C片段進(jìn)行基因克隆、重組表達(dá)、蛋白純化和免疫原性分析。應(yīng)用PCR技術(shù)直接從破傷風(fēng)梭菌64008菌株中擴(kuò)增出大小為1356bp的破傷風(fēng)毒素C片段基因,該片段是與靶細(xì)胞起結(jié)合作用的重鏈C端基因,經(jīng)DNA序列測定分析,擴(kuò)增出的基因與GenBank上登陸的序列AF154828的同源性達(dá)到99.2%。將此基因克隆入大腸桿菌融合表達(dá)載體pET-30a(+),構(gòu)建成重組表達(dá)質(zhì)粒pET-TetC,并在大腸桿菌Rosetta(DE3)中表達(dá),經(jīng)SDS-PAGE蛋白電泳鑒定,表達(dá)產(chǎn)物為50kD左右的特異性重組蛋白,重組蛋白的表達(dá)量占菌體總蛋白的33.5%,
[Abstract]:Tetanus is a serious spasmodic disease caused by tetanus toxin. The traditional toxoid vaccine is made of tetanus toxin which is detoxified by formaldehyde. There are still some problems: there are some side effects after inoculation, and Clostridium tetanus can form spores and is highly toxic. Production of vaccine is dangerous; formaldehyde treatment of toxoid is easy to cause pollution; chemical treatment of toxoid may be toxic reversal. Therefore, the existing toxoid vaccine needs further improvement and development, and the development of new genetic engineering vaccine is one of the directions. Based on the tetanus toxin C fragment gene, a highly expressed fusion protein was obtained by using E. coli expression system. Then the recombinant attenuated Salmonella typhimurium strain carrying tetanus toxin C fragment was constructed by using salmonella expression system. For the development of a new tetanus vaccine laid the foundation. 1. Cloning and expression of tetanus toxin C fragment in Escherichia coli Protein purification and immunogenicity analysis. PCR technique was used to amplify the tetanus toxin C fragment gene of 1356bp from Clostridium tetanus strain 64008. The fragment was a heavy chain C-terminal gene binding to target cells and was sequenced by DNA. The homology between the amplified gene and the AF154828 landing sequence on GenBank was 99.2%. The gene was cloned into Escherichia coli fusion expression vector pET-30a (), to construct a recombinant expression plasmid pET-TetC, and expressed in E. coli Rosetta (DE3). The expression product was identified by SDS-PAGE protein electrophoresis as a specific recombinant protein about 50kD. The expression of recombinant protein was 33. 5% of the total bacterial protein.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R392.1

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 單艷菊;破傷風(fēng)毒素C片段單克隆抗體的研制與鑒定[D];揚(yáng)州大學(xué);2007年

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本文編號：2263580