摘要
在传染性喉气管炎病毒（ＩＬＴＶ）北京Ｅ２株ＴＫ基因克隆、鉴定的基础上［１］，进一步构建转移载体质粒，为ＩＬＴＶＴＫ基因缺失毒株的构建作准备，先用ＰｓｔＩ将ｐＳＶｇａｌ线性化，再经ＥｃｏＲＩ不完全酶切，分离含ＳＶ４０立即早期启动子和增强子的４２Ｋｂ的ＬａｃＺ报告基因片段，并用Ｋｌｅｎｏｗ大片段补平，将克隆ＴＫ基因的载体ｐＴＫ用ＳｎａＢＩ处理，后用Ｔ４ＤＮＡ连接酶将以上两个片段平端连接成重组质粒，转化ＪＭ１０９，在Ｘｇａｌ、ＩＰＴＧ、Ａｍｐ、ＬＢ平板上筛选蓝色菌落，经琼脂糖凝胶电泳筛选到１２个阳性重组子，命名为ｐＰＴＫ，用ｐｓｔＩ和ＨｉｎｄＩＩ单酶切都得到了分子量为８１３Ｋｂ的质粒，进一步用狄高辛标记ＬａｃＺ基因片段作探针对ｐＰＴＫ进行打点杂交，结果均成阳性，证明了ＬａｃＺ基因已插入了ｐＴＫ，脂质体转染法也得到了蓝斑病毒，结果表明我们已成功构建了转移载体质粒ｐＰＴＫ。
BasedonthecloningandidentificationoftheTKgenefromavirulent(BeijingE2)strainofILTV.WesucceededinconstructinganinsertionvectortotransferthemarkergeneSV40LacZintotheILTVgenome.ThepsvgalplasmidcontainingthegenewithSV40immediateearlypromoterandenhancerLacZwasrestrictedwithPstI,thendigestedwithEcoRIincompletely,a4.2KbfragmentwhichhadSV40LacZcassetteseparatedwithlowmeltingpointagrose,andthecohesiveendsbluntedwithKlenowenzyme,PlasmidpTKwasrestrictedwithsinglecleavagesiteSnaBIsimultanously,TheSV40LacZcassetteandpTKplasmidwerebluntligatedwithT4DNAligaseyieldingarecombinant,designatedaspPTK,E.colistrainJM109wastransformedandgrownonagarplateinthepresenceofAmpXgalandIPTG,andbluecolonieswerepicked,usingelectrophresiswescreenedout12positiverecombinantplasmid,andwhenthepPTKwassubjectedonlytoPSTIorHindIIIrestriction,alinear8.13Kbband,equaltothelengthofSV40LacZpluspTKwasobtainedineachcase.FurtheridentificationwithdigoxigenlabelledLacZshowedthatpPTKwaspositive,Usinglipofectiontransfectionmethod,theresultingrecombinantwasusedwithvirulentparentstrainILTVtocotransfectchickenembryokidney(CEK)cells,yieldingaLacZlabelledrecombinantvirus.TheresultsindicatedthatwehadsuccessfullyconstructedthetransfervectorplasmidpPTK.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
1999年第3期262-266,共5页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
国家自然科学基金
广东省自然科学基金
