利用电穿孔法对狂犬病病毒srv9ψ区缺失株病毒的拯救及鉴定

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1、利用电穿孔法对狂犬病病毒SRV9W区缺失株病毒的拯救及鉴定毛丽萍魏玉圆王伟翟少华程瑶文兆海简子健新疆农业大学动物医学学院探讨电穿孔转染法对SRV9W区缺失株重组病毒的拯救及鉴定。利用电穿孔细胞转染法，将纯化的屯区缺失株pcDNA-NPMG-L全长质粒和pcDNA-N、pcDNA-P、pcDNA-M、pcDNA-G和pcDNA-L辅助表达质粒共转染至BHK-21细胞中，并进行盲传，借助RT-PCR、间接免疫荧光试验、Western-blot及透射电子显微镜对重组病毒进行鉴定。结果显示，重组病毒能够产生绿色荧光，透射电子显微

2、镜观察可见典型的弹状病毒，对重组毒株与亲本毒株G蛋內进行Western-blot验证，可获得相应目的条带。结果表明，通过电穿孔细胞转染法，能成功拯救出重组病毒SRV9区缺失株，为病毒的拯救提供新的转染方法，并能有效提高转染效率，为研发新型狂犬病疫苗提供参考。关键词：狂犬病病毒;电穿孔转染法;SRV9W区缺失株;拯救;同等贡献作者。基金：国家自然科学基金项目(31360623)RescueandIdentificationofRabiesVirusSRV9AeraDelectionstrainbyElectroporati

3、onTransfectionMAOLi-pingWEIYu-yuanWANGWei皿IShao-huaCHENGYaoWENZhao-haiTIANZi-jianCollegeofVeteiJji^vMedicine,XinjiangAgriculturalUniversity;Abstract：TheaimofthestudywastoinvestigatetherescueandidentificationofrecombinantvirusofSRV9withoutpseudogene屮regionviaelect

4、roporationtransfection.Thepurifieddeletedpseudogene^regionpCDNA-NPMG-LandthehelperplasmidspcDNA-N，pcDNA-P,pcDNA-M,pcDNA-GandpcDNA-Lwereco-transfectedintoBIIK-21cellsviaelectroporationtransfectionforvirusrescue,andtheBHK21cellswereusedforblindpassagesoftherecombin

5、antrabiesvirus.TherecombinantrabiesvirusSRV9deletedpseudogeneregionwasidentifiedbyRT-PCR,indirectimmunofluorescence,Western-blotandelectronmicroscopy.Theresultsshowedthattherecombinantviruscouldproducegreenfluorescenceintheindirectimmunofluorescenceassay,thetypic

6、alrhabdoviruswasobservedbytransmissionelectronmicroscopy.TherecombinantstrainandtheparentstrainGproteinweresubjectedtoWestern-blottoobtainthecorrespondingtargetband.Theaboveresultsshowedthattransfectionthroughelectroporationcellsinthisexperimentwasabletorescueits

7、recombinantvirusSRV9巾regiondeletionstrain.Thisstudyprovidedanewtransfectionmethodforrescueofvirus，andcaneffectivelyimprovethetransfectionefficiency,andprovideeffectivetheoreticalbasisandpracticalbasisforthedevelopmentofnewrabiesvirusvaccine.Keyword：Rabiesvirus;el

8、ectroporationtransfection;SRV9deletedpseudogene屮region;rescue;狂犬病(Rabies)是由狂犬病病毒引起的一种人畜共患的中枢神经系统传染病，病死率高达100%111。该病毒为弹状病毒科狂犬病毒属的单股负链RNA病毒。棊因组全长约12kb，由5个结构基因构成，