破伤风毒素C片段基因的克
发布时间：2018-10-11 08:51
【摘要】：破伤风是由破伤风毒素引起的一种严重的痉挛性疾病,有效的预防方法是接种破伤风类毒素疫苗。传统的类毒素疫苗是破伤风毒素经甲醛脱毒、精制而成的,现仍存在一些问题:接种类毒素后有一定的副反应发生;破伤风梭菌可形成芽孢形式,毒性高,生产疫苗有一定危险性;甲醛处理类毒素容易造成污染;化学处理后的类毒素可能发生毒性逆转。因此,现有的类毒素疫苗还需进一步改进和发展,开发新型的基因工程疫苗是方向之一。本研究以破伤风毒素C片段基因为基础,用大肠杆菌表达系统得到了高效表达的融合蛋白,之后又用沙门氏菌表达系统构建了运送破伤风毒素C片段的重组减毒鼠伤寒沙门氏菌菌株,为研制新型的破伤风疫苗奠定了基础。 1.破伤风毒素C片段基因的克隆及在大肠杆菌中的表达 本研究对破伤风毒素C片段进行基因克隆、重组表达、蛋白纯化和免疫原性分析。应用PCR技术直接从破伤风梭菌64008菌株中扩增出大小为1356bp的破伤风毒素C片段基因,该片段是与靶细胞起结合作用的重链C端基因,经DNA序列测定分析,扩增出的基因与GenBank上登陆的序列AF154828的同源性达到99.2%。将此基因克隆入大肠杆菌融合表达载体pET-30a(+),构建成重组表达质粒pET-TetC,并在大肠杆菌Rosetta(DE3)中表达,经SDS-PAGE蛋白电泳鉴定,表达产物为50kD左右的特异性重组蛋白,重组蛋白的表达量占菌体总蛋白的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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1
本文编号：2263581
[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.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392.1
【引证文献】
相关硕士学位论文 前1条
1 单艳菊;破伤风毒素C片段单克隆抗体的研制与鉴定[D];扬州大学;2007年
，本文编号：2263581
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