【作者】 王勇；

【导师】 宋方洲； 马永平；

【作者基本信息】 重庆医科大学 ， 分子生物学， 2006， 硕士

【摘要】 第一部分:pBV220-LTB基因克隆与表达目的:采用基因组提取方法从野生型产毒性大肠杆菌E.coli H 44815中提取LTB基因,利用基因工程技术将其重组于表达载体pBV220,然后对含有pBV220-LTB的工程菌进行表达,蛋白质分析,为研究该蛋白质的生物学特性提供基础。方法:用PCR方法扩增LTB目的基因片段,克隆至pBV220质粒中,构建pBV220-LTB的表达质粒,转化E.coli DH5α,经温度诱导表达重组蛋白。结果:经测序基因片段由374bp组成,为编码124个氨基酸残基的多肽。经SDS-PAGE分析相对分子量(Mr)约为30000D。结论: pBV220- LTB的原核表达质粒构建成功,并在大肠杆菌DH5α中高效的表达。第二部分:LTB蛋白质的家兔毒性实验目的:LTB的粘膜免疫作用已比较明确,但是任何疫苗的开发,安全性是必须考虑的问题,重组表达载体pBV220–LTB表达蛋白有无毒性,是其能否用于疫苗研究的前提,因此探讨重组LTB蛋白质对家兔的毒性作用,评价感染的可行性,为后续的双歧杆菌研究做准备,更多还原

【Abstract】 PARTⅠCloning and Expression of pBV220-LTBObjective: To construct and express pBV220-LTB,we distill Heat-labile enterotoxin B subunit from E.coli H 44815 by gene-engineering, which would lay a foundation for the biological idiosyncrasy.Methods: Heat-labile enterotoxin B subunit gene was amplified by PCR and cloned into plasmid pBV220. The recombinant plamsid was identified by sequencing ,then transformed into E.coli DH5a.. The transformant colony was induced with IPTG. Purify the expressed protein by Ni²⁺-NTA column chromatography. Expression of the protein was analyzed by SDS-PAGE .Results: The gene fragment at length of 396 bp was amplified and successfully cloned into plasmid pBV220.SDS-PAGE showed a protein band with relative molecular weight of 30 000,which was consistent with the expectation.Conclusion: The recombinant plasmid of pBV220-LTB was successfully constructed and Heat-labile enterotoxin B subunit gene was highly expressed in E.coli DH5a.PARTⅡThe rabbit`s toxicity experimentation of the protein of Heat-labile enterotoxin B subunit更多还原