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ID:36774451
大小:1.27 MB
页数:31页
时间:2019-05-15
《大肠杆菌不耐热肠毒素对新城疫疫苗的免疫增强作用研究》由会员上传分享,免费在线阅读,更多相关内容在学术论文-天天文库。
1、中国农业大学硕士学位论文大肠杆菌不耐热肠毒素对新城疫疫苗的免疫增强作用研究姓名:李月辉申请学位级别:硕士专业:预防兽医学指导教师:苏敬良20031101摘要本实验利用本课题组构建的表达LT突变体基因的质粒pLTR72<质粒的LT基因中编码第72位丙氨酸的密码子GCA被替换成为编码精氮酸的密码子AGA)和天然Lr质粒pLT分别转化大肠杆菌JMIOI,接种于LB培养基中培养,然后裂解菌体,纯化蛋白LTR72和1.T._I{j间接ELISA法检测目的蛋向ff勺表达情况。结果表明。LB培养基可蛆表达质粒pL7’R72和质粒DLT中的LT
2、突变体基因及LT基因。并可将产物分泌到大肠杆菌胞质中,成为具有GMI结合活性的突变体LT和天然LT。而JE-者在活性上无差异:将纯化蛋白LT和LTR72进行SDS—PAGE电泳,结果显示,纯化的突变体LT蛋白和天然LT蛋白的结构相似,都是由A、B二弧单位组成:在免肠袢结扎试验中,天然LT的毒性最大,突变体LT72的毒性很小,接近下阴性对照的水平。将不同剂量的单纯LTR72接种于1日龄健康雏鸡,结果显示,接种100pg范围内对鸡安全可靠,无不良反应,单纯LTR72免疫后对NDHI抗体效价无影响.但能椎特异性地刺激鸡体内淋巴细胞话性
3、增强。通过用不同剂量LTR72协同疫苗免疫后检测ND抗体的水平.确定疫苗中LTR72的最佳用量为25ug。将LTR72与NDLaSota疫苗混合后进行雏鸡免疫试验,结果表明,无论是高母源抗体免疫组.还是低母源抗体免疫组,LTR72一NO疫苗组的HI抗体效价均明显高于单纯疫苗组(P4、LT),突变体,免疫增强剂,鸡新城疫疫苗ImmuneEnhancementofEscherichiacoilHeat-labileEⅡterotoxiⅡonNewcastaldiseaseLaSotaStrainVaccinePostgraduate:LiYuehuiAdvhor:SuJingliangAbsWactThe2recombinantplasmidsD【丁R72andPLTweretransformedintoE.coliJMl01andthellculturedat37℃forabout24hoursingrowt5、hmediaLBrespectively.ThewholecelllysatesofalltheseculturedE.coliwerepreparedbythreecyclesoffrozen-thawandsonication600W×l0s×5untilallwel'ebrokencompletely.ThemutantsandLTwereexpressedfromthehOstOfJMlOlhambourmgtheplasmidspLTR72andpLTwereisolatedandpurifiedfi'omtheirw6、holecelllysaterespectivelybythemethodof(NH4)2S04precipitation,Gelfiltrationchromatograthy,D-galascosaelution.ThepurifiedproductsinthelysatesWeI'etestedbvGMl{LISA,theresultsshowedthatthemutantswereexpressedwellwhentheyweretransformedintotheE.coilJMl01strainandcultured7、intheLBmedia.1kresultsofGMl-ELISAshowedthatthemutantshadGMlbindingaetivity.InadditioIl,allthepooledsamplesweretested埘sDS-PAGE,andtheresunSshowedthatthemutantandLTconsistedoftwosubunits.ThemolecularWeightwereaboutl1.5KDaforE田and29KdaforLIA。Atlerthesampleswerlginjeeted8、iatethelumenoftheligatedilealloopoftherabbitisolatedwithligaturesfor24hours.fluidsecretionintothelumenwere5.4mlforLT,2.3mlforI积72an
4、LT),突变体,免疫增强剂,鸡新城疫疫苗ImmuneEnhancementofEscherichiacoilHeat-labileEⅡterotoxiⅡonNewcastaldiseaseLaSotaStrainVaccinePostgraduate:LiYuehuiAdvhor:SuJingliangAbsWactThe2recombinantplasmidsD【丁R72andPLTweretransformedintoE.coliJMl01andthellculturedat37℃forabout24hoursingrowt
5、hmediaLBrespectively.ThewholecelllysatesofalltheseculturedE.coliwerepreparedbythreecyclesoffrozen-thawandsonication600W×l0s×5untilallwel'ebrokencompletely.ThemutantsandLTwereexpressedfromthehOstOfJMlOlhambourmgtheplasmidspLTR72andpLTwereisolatedandpurifiedfi'omtheirw
6、holecelllysaterespectivelybythemethodof(NH4)2S04precipitation,Gelfiltrationchromatograthy,D-galascosaelution.ThepurifiedproductsinthelysatesWeI'etestedbvGMl{LISA,theresultsshowedthatthemutantswereexpressedwellwhentheyweretransformedintotheE.coilJMl01strainandcultured
7、intheLBmedia.1kresultsofGMl-ELISAshowedthatthemutantshadGMlbindingaetivity.InadditioIl,allthepooledsamplesweretested埘sDS-PAGE,andtheresunSshowedthatthemutantandLTconsistedoftwosubunits.ThemolecularWeightwereaboutl1.5KDaforE田and29KdaforLIA。Atlerthesampleswerlginjeeted
8、iatethelumenoftheligatedilealloopoftherabbitisolatedwithligaturesfor24hours.fluidsecretionintothelumenwere5.4mlforLT,2.3mlforI积72an
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