牛源无乳链球菌sip基因编码抗原表位序列的原核表达及其初步应用研究(1).docx
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内蒙古民族去学
硕士学位论文
牛源无乳链球菌Sip基因编码抗原表位序列的原核表达及其初步
应用研
姓名:
郎景民
申请学位级别:
硕士
专业:
预防兽医学
指导教师:
布日额
20100401
目的构建无乳链球菌Sip基因编码抗原表位序列的原核表达载体,在大肠杆菌中表达,并对其表达蛋白的免疫学活性及其应用进行研究。
方法提取无乳链球菌临床分离菌株基因组DNA,根据GenBank公布的无乳链球菌sip基因序列,应用DNAStar软件Protean程序预测sip基因抗原表位、亲水性和抗原性指数等参数,并设计引物,PCR扩增出sip抗原表位基因.将其插入至表达质粒pET-30(a)的多克隆位点(MCS),构建重组表达质粒sippET,以常规转化方法将sip-pET转入E.coli/BL21(DE3),IPTG诱导表达并优化诱导表达条件,应用SDS-PAGE电泳分析表达蛋白图谱,Western-blot技术验证重组蛋白的免疫活性。
运用Ni”亲和层析法在自然条件下纯化重组蛋白,并建立间接ELISA方法,应用该方法对无乳链球菌全菌体裂解免疫制剂免疫的家兔血清抗体滴度进行检测。
结果结果经测序和酶切分析表明,克隆获得了无乳链球菌sip抗原表位编码序列,且插入片段读码框架正确,原核表达重组质粒构建成功。
经IPTG诱导.BL2KDE3)/sip-pET系统表达出相对分子质量为40KD的可溶性融合蛋白(表达量占菌体总量的43.8%).Western-blot证实了重组蛋白的免疫活性。
纯化后蛋白含量为5.09mg/ml,间接ELISA方法检测3次免疫后58d的血清免疫组抗体滴度达到1:
3200,免疫70d后仍保持较高抗体滴度.
结论成功克隆牛源无乳链球菌sip抗原表位序列,该序列897bp,编码299个氨基酸残基,构建的重组表达载体在大肠杆菌中获得高效可溶性表达,并建立了检测无乳链球菌免疫制剂免疫抗体滴度的间接ELISA方法,为进一步研究SIP抗原表位重组蛋白免疫原性及无乳链球菌疫苗免疫效果研究奠定了基础。
关键词:
牛源无乳链球菌;sip基因;原核表达;纯化:
间接ELISA;抗体检测
StudyontheProkaryoticexpressionandPreliminary
ApplicationofCodingRegionGeneofSipEpitopeof
StreptococcusagalactiaeFromBovine
Abstract
Objectivlbconstructprokaryoticexpressionvectorofsipgene,expressitinEscherichiacoliBL21,andstudytheimmunocompetenceandapplication.
MethodsGenomicDNAwasextractedfromstreptococcusagalactiae,epitopesites,hydrophilicityandantigenicindexparameterswerepredictedbyDNAStarProteanprogramaccordingtotherelevantnucleotidesequencefromGenBank^andapairofspecificprimerswasdesignedtoamplifytheepitopeofsipgenebyPCR.Thefragmentwasclonedintothemultiplecloningsite(MCS)ofpET»30(a),fbnningtherecombinantprokaryoticexpressionvector(sip-pET).Therecombinantplasmidsip-pETwastransformedintoE.coliBL21(DE3)bycommonmethodandtheBL21(DE3)/pETwasinducedbyIsopropyl-p-D-thioglactoside(IPIG)toexpressfusionprotein,optimizetheexpressioncondition.ThentheexpressedproteinsinE.coliBL21(DE3)wereanalyzedwithSDS-PAGEelectrophoresis.WesternblottingwasperformedtoidentifytheImmuneactivefusionprotein.Therecombinantproteinwaspurifiedwithimmobilizednickeionaffinitychromatography,andanindirectenzymelinkedimmunosorbentassaywasdeveloped.Takingthismethodtotesttheserumtitersofvaccinatedrabbitswhichwereimmunizedwithimmunepharmaceuticsofstreptococcusagalactiae.
ResultsTherecombinantplasmid(sip-pET)wasobtainedanditsopeningreadfram^ORF)wasverifiedbysequenceanalysis.TheresultsofSDS-PAGEshowedthattherewasanovalproteinband,whichwas40KD.Westemblottingassayprovedthatthefusionproteincouldbespecificallyrecognizedbyanti-Str.agalactiaeantibody.ThedensityofSipproteinwas5.09mg/mlafterpurification,theantibodyvalenceofstreptococcusagalactiaewere1:
3200whentestedbyindirectenzymelinkedimmunosorbentassay,Specificantibodycouldbedetectedin70daysafterinjectionimmunization.
ConclusionsTheseresultsshowedthatcodingregiongeneofsipepitopeofstreptococcusagalactiaefrombovinewassuccessfullycloned,thesequencewascomposedof897nucleotidesencodingapolypetideof299aminoacids,prokaryoticexpressionvectorwasefficientlyexpressedinE.coliBL21(DE3)andanindirectenzymelinkedimmunosorbentassayfordetectionofBovineMastitisStreptococcusagalactiaewasdeveloped.Thepreviousworkhasnotonlyrolloutabasisforthestudiesofantigenicityidentificationoftherecombinantprotein,butalsolaidanecessaryfoundationforfutureresearchonpossibleImmuneeffectofstreptococcusagalactiaeinthefuture.
Keywords:
BovinesourceStreptococcusagalactiae;sipgene;Prokaryoticexpression:
Purification;
Indirectenzymelinkedimmunosorbentassay
Directedby:
prof.BuRie(Ph.D)
AppIicantforMasterdegree:
LANGJingmin(preventiveveterinaryScience)
(CollegeofAnimalScienceandTechnology,!
nnerMongoliaUniversityfbrNationalities,Tongliao028043,China)
英文缩写词表
英文缩写
英文名称
中文名称
4«CN
4-Chloro1-naphthol
四氯奈酚
A492
Absorptionvalueat492nm
492纳米处的吸光值
Amp
Ampicillin
氨千青霉素
APS
Ammoniumpersulfate
过硫酸铉
bp
Basepair
碱基对
CP
Caspsularpolysaccharide
荚膜多糖
CSF
Colony-stimulatingfactor
集落刺激因子
DNA
deoxyribosenucleicacid
脱氧核糖核酸
dNTP
Deoxynucleosidetriphosphate
脱氧核吾三磷酸
DTT
Dithiothreitol
_硫苏糖醇
E.coli
Escherichiacoli
大肠杆菌
E.coliBL2I
EscherichiacoliBL21
基因「•程表达菌
E.coliJM109
EscherichiacoliJM109
基因1程克隆菌
EB
EthidiumBromide
漠化乙锭
ED1A
ethylenediaminetetraaceticacid
乙一胺四乙酸
ELISA
Enzyme-linkedimmunosorbentassay
酶联免疫吸附实验
EP
Eppendorf
离心管
FCA
Freundcompleteadjuvant
福氏完全佐剂
FIA
Freundincompleteadjuvant
福氏不完全佐剂
Genebank
Genebank
核吾酸序列数据库
h
Hour
小时
HRP
Horseradishperoxidase
辣根过氧化物酶
IF
Interferon
干扰素
Ig
Immunoglobulin
免疫球蛋白
IL
Interleukin
白细胞介素
IPTG
isopropylthio-p-D-thiogalactoside
异丙基■硫代半乳糖昔
Kan
kanamycin
卡那霉素
KD
kilodalton
千道尔顿
L
Liter
升
LB
Luria-Benrtanimedium
LB培养基