【作者】 刘婷；

【导师】 焦新安；

【作者基本信息】 扬州大学 ， 遗传学， 2005， 硕士

【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%,更多还原

【Abstract】 Tetanus is a severe spastic disease caused by tetanus toxin, which can be prevented by immunization with tetanus toxoid. There are some disadvantages in traditional tetanus vaccine based on chemical inactivated tetanus toxin by formaldehyde such as side effects, dangerous brought by the spore, pollution made by formaldehyde and the reversion of virulence. So current tetanus vaccine should be improved, especially needed to develop genetic engineering vaccines. In this study tetanus toxin fragment C was highly expressed in E.coli expression system and a recombinant attenuated Salmonella typhimurium strain expressing tetanus toxin fragment C was constructed, and the immunobiological properties of the expressed protein was evaluated, all of these provide some foundation for further studying of tetanus vaccine.1. Cloning, expression of tetanus toxin fragment C gene in E.coliThe gene of tetanus toxin fragment C (1356bps) was amplified from Clostridium tetani strain 64008 by PCR. The PCR product was inserted into the high-expression vector pET-30a(+) for sequencing and expressed in Exoli Rosetta(DE3). It was shown that sequence has 99.2% homogeneity to that in GenBank. The expressed protein was purified by His Bind Resin Kit. The result of SDS-PAGE verified that a desired recombinant protein with molecular weight of 50kD was expressed and accounted for33.5% of the total bacterial protein. Western-blot analysis further indicated this expressed product had the immunogenicity of tetanus toxin fragment C. The expressed protein initiated enough antibodies in immunized mice that were able to protect mice against a challenge with tetanus toxin.2. Construction and identification of the attenuated Salmonellatyphimurium strain expressing tetanus toxin fragment CTo construct a recombinant attenuated Salmonella typhimurium strain which expresses tetanus toxin fragment C, the gene fragment encoding for tetanus toxin fragment C has been cloned into the expression vector pYA3333 containing asd gene, generated a new recombinant plasmid pYA3333-TetC, which was transformed into Exoli X6212 with asd gene deletion. The recombinant plasmid pYA3333-TetC was further transformed into the host cells by electroporation, yielding the live vaccine strain X4550(pYA3333-TetC), which is a balanced lethal recombinant. Stability and growth curve of the recombinant strain was determined. The results showed that recombinant X4550(pYA3333-TetC) maintained good stability after 100 passages in vitro and the growth status of X4550(pYA3333-TetC) as same as that of X4550(pYA3333). The recombinant strain X4550(pYA3333-TetC) was safe and could provide significant protection against a challenge with tetanus toxin in immunized mice. This study lays the preliminary foundation for the construction of a novel tetanus vaccine.更多还原