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1、猪白细胞介素．2基因的克隆及其重组禽痘病毒载体的构建nCT^llORICULTURAEBOREILI-SIHICa华北农?2011,26(5):71—75猪白细胞介素.2基因的克隆及其重组禽痘病毒载体的构建王淑娟,陈红英,魏战勇,刘金朋,耿静微,崔保安(河南农业大学牧医工程学院,河南郑州450002)摘要:采用RT—PCR技术自豫南黑猪脾淋巴细胞中扩增猪儿一2基因(p/L一2),RT—PCR产物进行T—A克隆并测序,获得了猪儿一2基因序列.将BamH1酶切的猪1L一2基因非定向克隆到BamH1酶切并去磷酸化的pSY538载体上,通过PCR,酶切和测序鉴定,筛
2、选正向插入的重组质粒pSY538/plL一2.NotI酶切pSY538/plL一2后,得到含有双启动子LP2和EP2的猪/L一2基因片段,将其亚克隆到含有LacZ基因的pSY681载体上,筛选正向插入的重组禽痘病毒转移质粒pSY681/plL一2.测序结果表明,克隆的豫南黑猪/L一2基因长482bp,包含1个完整读码框(465bp),与GenBank中其他5条猪/L.2基因核苷酸同源性为99.9%.重组质粒pSY681/pIL一2经酶切,测序鉴定,证实含有目的片段,且连接,构建正确.成功构建了重组禽痘病毒转移质粒pSY681/pIL一2,为猪IL一2生物学功
3、能的进一步研究和开发奠定基础.关键词:猪白细胞介素一2;克隆;重组禽痘病毒;载体;构建中图分类号:$828.2;Q782文献标识码:A文章编号:1000—7091(2011)O5—0071—05CloningofPorcineIL-2GeneandItsConstructionofRecombinantFowlpoxVirusesVectorWANGShu—juan,CHENHong—ying,WEIZhan—yong,LIUJin—peng,GENGJing—wei,CUIBao—an(CollegeofAnimalHusbandryandVeterina
4、ry,HenanAgriculturalUniversity,Zhengzhou450002,China)Abstract:Porcine/L一2genewasamplifiedbyRT-PCRfromthetotalRNAextractedfromthesplenocyteofYunanpig.ThePCRproductwasclonedandsequenced.TheporcineIL一2(p/L一2)genewasdigestedbyBamHIandclonedintopSY538vectorwhichwasdigestedbyBamHIanddeph
5、osphorizedbycalfalkalinephosphatase.BymeansofthePCR,restrictionenzymedigestionandsequencing,therecombinantplasmidpSY538/plL一2wasselect—ed.AfterpSY538/plL一2wasdigestedbyNotI,theplL一2fragmentcontainingLP2andEP2wasachievedandsub—clonedintopSY681vectercontainingLacZgene,thentherecombin
6、antplasmidpSY681/plL一2wasdeveloped.Theresultsindicatedthattheclonedp/L一2genehad482bp,includingoneORF(465bp).The/L一2genesequenceofYunanpighad99.9%nucleotidehomologywiththe5pig儿一2genespublishedinGenBank.EnzymedigestionandDNAsequencingresultsconfirmedthatthesequenceoftherecombinantpla
7、smidpSY681/plL一2containedthetargetfragment,andtheligationpartwascorrect.TheresultsshowedthattherecombinantplasmidpSY681/plL一2wasconstructedcorrectly,pavingthewayforfurtherstudyofbiologicalfunctionandfurtherapplicationofporcineIL-2.Keywords:PorcineIL?2;Clone;Recombinantfowlpoxvirus;
8、Vector;Construction白细胞介素一2
