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1、猪FMDV受体B3亚基配体结合域的基因克隆及其多克隆抗体制备DOC格式论文,方便您的复制修改删减猪FMDV受体B3亚基配体结合域的基因克隆及其多克隆抗体制备(作者:单位:邮编:)作者:独军政,高闪电,常惠芸,丛国正,林彤,邵军军,刘湘涛,才学鹏【摘耍】冃的:克隆猪FMDV受体B3亚基的配体结合域(LBD)基因,利用原核细胞表达B3亚基的LBD片段,纯化目的蛋白后制备兔多克隆抗体。方法:利用RT-PCR方法从FMDV实验感染猪的肺组织中克隆B3亚基的LBD基因,将其与pGEMT-easy载体相连构建pGEM/B3LBD,经BamHI/XhoI酶切后回收B3LB
2、D片段,与同样酶切处理的原核表达载体pGEX4T・1相连,构建原核表达质粒pGEX/P3LBD,将其转化感受态细胞BL21(DE3)Z以IPTG诱导表达B3LBD蛋白。纯化目的蛋白后免疫新西兰白兔制备多克隆抗体。通过ELISA和Westernblot鉴定血清效价和特异性。结果:猪B3亚基的LBD基因含有507个核甘酸,编码169个氨基酸,猪B3LBD基因与牛、人、黑猩猩、猫猴、马、犬、挪威大鼠、家鼠、和鸡的B3LBD基因核昔酸序列同源性分别为90.3%、92.3%、92.1%、DOC格式论文,方便您的复制修改删减91.3%>90.5%、90.3%、87.8%
3、、85.2%、79.5%。哺乳动物的B3亚基LBD基因同源性较高,与鸡的同源性较低。实现了重组猪B3LBD蛋口在大肠杆菌屮的高效表达,SDS-PAGE显示其相对分子质量(Mr)约为44000,制备的兔多克隆抗体效价达1:12800以上。结论:实现了猪FMDV受体B3LBD的基因克隆、原核表达和多克隆抗体的制备,为深入研究受体03亚基在FMDV感染过程屮的作用机制奠定了基础。【关键词】FMDV病毒受体猪B3LBD基因原核表达多克隆抗体[Abstract]AIM:Tocloneandexpresstheligandbindingdomain(LBD)cDNAof
4、porcineintegrinP3asfoot-and-mouthdiseasevirus(FMDV)receptorandprepareitspolyclonalantibody.METHODS:TheLBDcDNAofporcine03wasobtainedfromthelungtissueofpiginfectedwithFMDVbyRT-PCR,andtherecombinantplasmidpGEM/B3LBDwasconstructed.AfterdigestedwithBamHI/XhoI,theB3LBDfragmentwassubclone
5、dintoprokaryoticexpressionvectorpGEX4T-1・TherecombinantexpressionplasmidpGEX/P3LBDwasconstructedandtransformedintoE.coliBL21(DE3).Therecombinantporcinep3LBDproteinwasexpressedafterIPTGinductionandpurifiedfromtotalproteinofBL21(DE3).TherabbitsfromNewZealandwereimmunizedwiththepurifi
6、edfusionproteintopreparepolyclonalantibody,whichwasidentifiedbyWesternblotandELISA・DOC格式论文,方便您的复制修改删减RESULTS:The507bpcDNAofporcine[33LBDencodedapolypeptideof169aminoacids.Thesimilarityofnucleotidesequeneep3LBDbetweenpigsandcattle,humanbeing,chimpanzees,rhesusmonkeys,horses,dogs,Nor
7、wayrats,mice,chickenswas90.3%,92.3%,92.1%,91.3%,90.5%,90.3%,87.8%,85.2%,79.5%,respectively.Thep3LBDgeneofmammalsexhibitedhighsequeneehomology・Therecombinantp3LBDproteinwasexpressedefficientlyasinclusionbodyafterIPTGinductionandwasapproximately44000.Thetiterofthepolyclonalantibodyag
8、ainstthepurifiedB3LBDprote