表达鸡Ⅱ型干扰素基因的重组鸡痘病毒的构建  

Construction of Recombinant Fowlpox Virus Expressing Chicken Type Ⅱ Interferon

作　　者：程坚[1] 刘秀梵[1] 彭大新[1] 吴艳涛[1] 

机构地区：[1]扬州大学畜禽传染病学农业部重点开放实验室,扬州225009

出　　处：《农业生物技术学报》2002年第2期152-155,共4页Journal of Agricultural Biotechnology

摘　　要：以插入鸡Ⅱ型干扰素(IFN-Ⅱ)cDNA序列的重组质粒AIFN为模板,根据鸡痘病毒早晚期启动子PE/L的序列设计上游引物,鸡IFN-Ⅱ基因的cDNA3'端序列设计下游引物,采用PCR法扩增包括启动子PE/L和鸡IFN-Ⅱ全长cDNA序列的基因片段。将该扩增片段定向插入鸡痘病毒转移载体1175中,获重组转移载体1175IFN,用以转染已感染鸡痘病毒282E4疫苗株(wt-FPV)的鸡(Gallusgallus)胚成纤维细胞(CEF)。1175IFN与wt-FPV基因组DNA之间的同源重组产生了表达IFN-Ⅱ的重组鸡痘病毒rFPV-IFN-Ⅱ。通过在含X-gal的营养琼脂上连续挑选蓝色病毒蚀斑获得并纯化rFPV-IFN-Ⅱ。以细胞病变抑制实验证实,rFPV-IFN-Ⅱ感染的CEF表达了具有抗水泡性口炎病毒(VSV)活性的鸡IFN-Ⅱ。rFPV-IFN-Ⅱ(M.O.I=0.01)感染CEF72h后,培养上清中IFN-Ⅱ的表达量为1280U/mL。动物实验表明,rFPV-IFN-Ⅱ接种雏鸡7d或14d后,其在体内表达的IFN-Ⅱ可显著减轻载体鸡痘病毒对1日龄SPF雏鸡体重增长的抑制作用。Accordingto thenucleotidesequenceof thepromoterPE/Landthenucleotidesequenceatthe3'-terminalof chicken typeⅡinterferon(IFN-Ⅱ)gene,a pairof primersweredesignedandusedto amplifythechickenIFN-Ⅱgenewithitsupstream promoterPE/LfromtherecombinantplasmidAIFN.Thentheamplifiedfragmentwas insertedintothetransferringvector1175,resultinginrecombinanttransferringvector1175IFN,whichwaslaterusedto transfectthechicken(Gallus gallus) embryofibroblasts(CEF)pre-infectedwithwidetypefowlpoxvirusto generaterecombinantfowlpoxvirusexpressingchickentypeⅡinterferon(rFPV-IFN-Ⅱ).rFPV-IFN-Ⅱwasobtainedandpurifiedby selectionof blueplaqueson theCEFoverlaidwithagarcontainingX-gal.In vitro,IFN-Ⅱexpressedby rFPV-IFN-ⅡinfectedCEFsuppressedthecytopathiceffect(CPE)inducedby vesicularstomatitisvirus(VSV)andthetiterof expressedIFN-Ⅱin theculturesupernatantof therFPV-IFN-ⅡinfectedCEFwas1280U/mL72h post inoculation.In vivo,theexpressedIFN-Ⅱby rFPV-IFN-Ⅱsignificantlyreducedthebodyweightlossof chickensdueto vaccination stressof FPV.

关 键 词：表达 鸡Ⅱ型干扰素基因 重组鸡痘病毒 基因工程苗 

分 类 号：S858.315.3[农业科学—临床兽医学] S852.65[农业科学—兽医学]