预防仔猪腹泻基因工程菌株的构建

【作者】 柏佳宁；

【导师】 赵宝华；

【作者基本信息】 河北师范大学 ， 遗传学， 2006， 硕士

【摘要】 由产肠毒素型大肠杆菌引起的仔猪黄白痢和由魏氏梭菌引起的仔猪红痢是影响猪体健康的常见疾病,可严重影响仔猪的存活率和出栏率,给养猪业造成了巨大的经济损失。 本文采用微生物分离方法从栾城县某养猪场患病仔猪的粪便中分离到致病性大肠杆菌,经形态学、生化和分子生物学鉴定为产肠毒素型大肠杆菌。以此菌株为研究对象,根据GeneBank公布的猪源致病性大肠杆菌LT_B基因全序列设计了一对引物,以分离菌株的基因组为模板扩增出LT_B基因,序列分析表明与其它参考菌株M17873（猪源ETEC的LT_B）序列同源性为100%。然后,利用定点突变技术将形成ST₁分子内二硫键的半胱氨酸碱基进行了突变,最后将突变的ST₁基因与LT_B基因融合,定向克隆于原核表达载体pET-28b中,得到了重组质粒pBETST₃LT_B,转化受体菌BL21（DE3）,得到重组菌株BL21（DE3）（pBETST₃LT_B）,该菌株经IPTG诱导后,SDS-PAGE分析可见其表达的30KD特异蛋白条带,经ST和LT ELISA检测试剂盒检测,重组菌株表达的ST₁-LT_B融合蛋白能够被ST和LT单抗识别。从本实验室保存的预防仔猪红痢的基因工程候选菌株的质粒中扩增出魏氏梭菌α-β毒素保护性抗原基因,通过分子生物学手段构建到ST₁-LT_B基因下游,得到了重组菌株BL21（DE3）（pBETST₃LT_Bαβ）,该菌株经IPTG诱导后,SDS-PAGE分析可见其表达的110KD特异蛋白条带。将构建的基因工程菌株BL21（DE3）（pBETST₃LT_B）和BL21（DE3）（pBETST₃LT_Bαβ）灭活后分别辅以中草药佐剂进行了初步免疫原性研究,结果显示本实验构建的2株基因工程菌株均具有较好的免疫保护作用,为进一步研制预防仔猪腹泻的基因工程疫苗奠定了坚实的基础。更多还原

【Abstract】 Yellow and white scour of newborn piglets caused by pathogenic Escherichia.coli and red scour caused by Clostridium Perfringens severely influence the livability as well as the growth of newborn piglets and result in tremendous economic loss in stockbreeding.Pathogenic E.coli was isolated from feces of sick newborn piglets in Luancheng hoggery and was identified as intestinal origin through the methods of morphology, biochemistry and molecular biology. A pair of primers was designed according to the full nucleotide sequence of LT_B gene of swine pathogenic Escherichia.coli published by genebank. LTb gene was amplified with the isolated strain’s genome as the template. Alignment with other LTb genes published in NCBI suggested that the homogeneity with M17873 is 100%. Codons encoding cystines forming ST₁ internal disulfide bonds were mutated by gene mutation technology. The mutated ST₁ gene was fused with LT_b and inserted to prokaryotic expression vector pET-28b by directional clone. SDS-PAGE demonstrated that a specific band of 30KDa was expressed by recombinant BL21 （DE3） （pBETST₃LT_B） induced by IPTG The fusion protein ST₁-LT_B could be recognized by anti-ST and anti-LT monoantibodies by E1ISA analysis. The protective antigen, α-β toxin gene of Clostridium Perfringens was amplified from the plasmid of candidate strain for the prevention of red scour of newborn piglets and inserted to the downstream of ST₁-LT_b gene. The recombinant BL21 （DE3） （pBETST₃LT_Bαβ） expressed a recombinant protein of 110KDa by the induction of IPTG Pilot study on the inactivated vaccines BL21 （DE3 ） （pBETST₃LT_B） and BL21 （DE3） （pBETST₃LT_Bαβ） with Chinese medicine as adjuvant showed that these kind vaccines have protective ability, which establishes a solid basis for the further study on the genetic engineering vaccines against scour of newborn piglets.更多还原