【作者】 罗维；

【导师】 余兴龙；

【作者基本信息】 湖南农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 1、产肠毒素性E．coliLT全基因的克隆与序列分析根据GenBank中收录的LT基因序列，设计并合成一对引物，以猪产肠毒素大肠杆菌临床分离株ECOH1的DNA为模板，通过PCR技术扩增出约1.3kb的片段。将此PCR产物克隆到pMD18-T载体上，构建了重组质粒pMDTLT，测序结果表明所克隆的LT基因序列全长为1334bp，与GenBank收录的序列的核苷酸同源性在97.6％-98.8％之间。2、E．coli野生株F18菌毛FedF基因的克隆表达及免疫原性研究根据Z26520中fedF序列设计1对引物，从发病仔猪的腹泻粪便分离获得的大肠杆菌中扩增得到fedF基因，经纯化、连接、转化，将其克隆到pMD18-T中，所克隆fedF基因序列与GenBank中收录的其他fedF基因序列的同源性在97.8％～99.2％。另设计1对引物，利用基因工程方法将所克隆的fedF的编码序列亚克隆到表达载体pBV220中，转化大肠杆菌BL21(DE3)LysS获得表达重组菌。SDS-PAGE电泳及Western-blotting分析表明重组菌在诱导后可以表达特异性的FedF蛋白，该重组蛋白分子量约为30kDa。该重组蛋白的表达量占菌体总蛋白量的11.17％。将纯化的重组FedF蛋白免疫小鼠，攻毒试验表明免疫小鼠可完全抵抗致死性的E．coli的感染。3、大肠杆菌LTB-FedF融合蛋白的表达及免疫原性研究经PCR、酶切、连接和转化将LTB成熟肽基因按预定的阅读框架插入到表达质粒pET28a(+)中，获得重组质粒pETLTB，用同样的方法将fedF成熟肽基因片段插入到重组质粒pPLTB中LTB基因的3’末端，获得LTB-fedF融合基因的质粒pETLTBFedF。SDS-PAGE与Western-blotting分析表明转化该质粒的重组大肠杆菌在IPTG诱导下可以高效表达融合蛋白LTB-FedF，该重组蛋白分子量约40kDa。该融合蛋白的表达量占菌体总蛋白量的23.17％。融合蛋白经纯化后免疫小鼠，攻毒保护试验表明免疫小鼠只得到部分保护。结果说明在此融合蛋白中LTB起到了免疫抑制作用，其原因有待进一步的研究。更多还原

【Abstract】 1. Cloning and Sequence Analysis of LT Gene of EnterotoxigenicE.coliOne pair of primers was designed according to LT sequences published in Genbankto amplify LT gene by PCR from a clinic isolated Escherichia coli strain containingthe LT gene. The PCR product was cloned into pMD18-T vector, the recombinantplasmid was named pMDTLT. The cloned LT gene was sequenced. Results ofhomology comparison showed that LT gene cloned in this study share 97.6 % to 98.8% identity with the LT gene published on GenBank in nucleotide sequence level.2. Expression and immunogenic study of E.coli F18 fimbriae FedFproteinAccording to the fedF gene sequence of F18 accessed on GenBank, a pair of primerswas designed. Then fedF gene products were amplified from field strain isolated frompiglet suffered from diarrhea. After purification, ligation and transformation, fedFgene was successfully cloned into pMD18-T, the cloned gene was confirmed to have97.8%～99.2% identity in gene sequence level with other fedF gene in GenBank.Then the cloned fedF gene was subsequently sub-cloned into expressing vectorpBV220 by using another set of primers via biological engineering’methods and theresulting recombinant plasmid was then transformed into BL21(DE3) LysS. bacteria.It was confirmed by SDS-PAGE analysis and Western-blotting that the recombinantBL21 bacteria induced by IPTG were able to express the FedF protein with moleculeweight of 30kDa. The induced FedF protein account 11.17% of total bacterial protein.Mice were immunized with purified FedF protein, and the immunized mice allprotected from lethal E.coli strain challenge.3. Expression and immunogenic study of E.coli LTB-FedF fusionproteinThe gene coding for the LTB without signal peptide cloned into plasmid vectorpET28a(+) named pETLTB by genetic engineering methods. The 5’ terminus of thegene that code for F18 fedF was genetically fused to the 3’ terminus of the genecoding for the LTB, resulted in a recombinant plasmid pETLTBFedF which was wastransformed into E.coli BL21(DE3) LysS. The recombinant bacteria containingpETLTBFedF was able to express large quantities of fusion protein (LTB-FedF) underinduction of IPTG, the amount of the induced fusion protein whose expected moleculeweight was about 40kDa account for23.17% of total bacterial protein. The LTB-FedFprotein was further confirmed by SDS-PAGE analysis and Western-blotting. Theinduced LTB-FedF protein was purified and immunized into 6 week mice, and theimmunized mice showed partial protection to lethal challenge of E.coli strain, whichsuggest the LTB exhibited suppression on immune system, further study of LTB isnecessary to explain this phenomena.更多还原