停乳链球菌内蒙古分离株MIG基因的克隆和表达.pdf

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1、分类号S852.62学校代码10129UDC11.220学号08201071停乳链球菌内蒙古分离株MIG基因的克隆和表达CloningandProkaryoticExpressionofMIGGeneSequenceofStreptococcusdysgalactiaefromStrainInnerMongolia申请人：刘金晔学科门类：农学学科专业：预防兽医学研究方向：微生物与免疫指导教师：郝永清教授论文提交日期：二〇一一年五月摘要停乳链球菌是引起奶牛乳房炎的主要病原菌，根据GenBank中登陆的停乳链球菌表面蛋白MIG基因序列，设计一

2、对引物，采用PCR的方法从临床分离的停乳链球菌内蒙分离株基因组DNA中扩增出MIG基因，得到一条约1900bp的片段。将其连入PMD-19T载体中，经酶切，PCR及序列测定法进行鉴定。结果表明，MIG基因与GenBank中停乳链球菌MIG基因(AF354651)序列的同源性为95%。构建原核表达载体PET-32ａ(+)-MIG，随后转化到大肠杆菌BL21(DH5a)中表达。表达产物进行SDS-PAGE分析后，表达的重组蛋白大小为89ku。将纯化的MIG重组蛋白免疫家兔，用ELISA检测其血清抗体。用制备出的抗血清与链球菌全菌进行玻片凝集试

3、验，出现凝集颗粒。结果表明MIG重组蛋白具有较强的免疫原性，为停乳链球菌疫苗的研制奠定基础。关键词：乳房炎停乳链球菌；MIG蛋白；克隆；原核表达；抗血清CloningandProkaryoticExpressionofMIGGeneSequenceofStreptococcusdysgalactiaefromStrainInnerMongoliaAbstractStreptococcusdysgalactiaeisthemajorpathogensofmastitisindairycows,S.dysgalactiaewerederive

4、dfromthemastitisBovineinsomeareasofInnerMongolia。ApairofprimerswasdesignedaccordingtoStreptococcusdysgalactiaeMIGsurfaceproteingenesequencesinGenBank.Subsequently,theMIGgeneof1900bpwasamplifiedbyPCR.Then,thegenewasclonedintoPMD-19Tvecto，andidentifiedbydigestionwithrestric

5、tionendonucleases,PCRandsequencing.TheDNAsequenceanalysisconfirmedStreptococcusdysgalactiaeinGenBank,MIGgene(AF354651)sequencehomologyof95%.AporkaryoticexpressionpET-32a(+)-MIGwasconstructed.TherecombinantplasmidwastransformedintoBL21(DH5a)/pETsystemforexpression.Therecom

6、binantproteinwasanalyzedbySDS-PAGE，ithadanapproximatemolecularmassof89ku.Withthepurifiedproteinimmunerabbits,andanti-MIGantibodiesweremeasuredbyELISA.WiththeantiserumpreparedwithwholebacteriaStreptococcusslideagglutinationtest,agglutinationparticlesappeared.Theresultsshow

7、thattheMIGfusionproteinwithstrongimmunogenicityforvaccinedysgalactiaebasis.Keywords:Streptococcusdysgalactiaemastitis;MIGprotein;cloning;Prokaryoticexpression;Anti-serumDirectedby:Prof.HAOYongqing（Ph.D）ApplicantforMasterdegree:LIUJinye（VeterinarianMicrobiology)(Collegeofv

8、eterinaryMedicine.InnerMongoliaAgriculturalUniversity.Hohhot010018.China)目录1引言..................