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《鴨白介素_18基因重組真核表達(dá)載體的構(gòu)建及其表達(dá)產(chǎn)物的生物學(xué)活性》由會(huì)員上傳分享，免費(fèi)在線閱讀，更多相關(guān)內(nèi)容在教育資源-天天文庫(kù)。
1、生物工程學(xué)報(bào)ChinJBiotech2008,September25;24(9):1568-1572journals.im.ac.cnChineseJournalofBiotechnologyISSN1000-3061cjb@im.ac.cn?2008InstituteofMicrobiology,CAS&CSM,Allrightsreserved研究報(bào)告鴨白介素-18基因重組真核表達(dá)載體的構(gòu)建及其表達(dá)產(chǎn)物的生物學(xué)活性1,211,2111陳紅英,李新生,崔保安,夏平安,張紅英,楊明凡1河南農(nóng)業(yè)大學(xué)牧醫(yī)工程學(xué)院,鄭州4500022河南省動(dòng)物性食品安全重點(diǎn)實(shí)驗(yàn)室,鄭州450002摘要:從
2、克隆質(zhì)粒pGEM-DuIL-18擴(kuò)增出鴨IL-18全基因片段,克隆到真核表達(dá)載體pcDNA3.1(+)中,構(gòu)建重組質(zhì)粒pcDNA3.1/DuIL-18(簡(jiǎn)稱pDuIL-18)。將重組質(zhì)粒pDuIL-18轉(zhuǎn)染Cos7細(xì)胞,轉(zhuǎn)染細(xì)胞中含鴨IL-18基因的mRNA。SDS-PAGE分析表明,表達(dá)產(chǎn)物是與鴨IL-18相符的約23000的蛋白條帶。鴨淋巴細(xì)胞轉(zhuǎn)化試驗(yàn)表明,表達(dá)產(chǎn)物對(duì)鴨淋巴細(xì)胞具有明顯誘導(dǎo)轉(zhuǎn)化作用。重組質(zhì)粒pDuIL-18對(duì)H9亞型禽流感滅活疫苗免疫增強(qiáng)作用的研究表明重組質(zhì)粒pDuIL-18能夠提高禽流感滅活疫苗誘發(fā)的細(xì)胞免疫應(yīng)答,為研究能夠更好地防制禽流感的新型疫苗提供了新的思
3、路。關(guān)鍵詞:白介素18(IL-18),鴨,真核表達(dá),淋巴細(xì)胞轉(zhuǎn)化試驗(yàn),HI試驗(yàn)ConstructionofEukaryoticExpressVectorofDuckInterleukin18GeneandIdentificationofBioactivityofItsExpressedProtein1,211,2111HongyingChen,XinshengLi,BaoanCui,Ping’anXia,HongyingZhang,andMingfanYang1TheCollegeofAnimalHusbandryAndVeterinary,He’nanAgriculturalUni
4、versity,Zhengzhou450002,China2AnimalFoodSafetyKeyLaboratory,He’nanProvince,Zhengzhou450002,ChinaAbstract:DuckIL-18genewasamplifiedfromplasmidpGEM-DuIL-18byPCR.ThePCRproductdigestedwithPstIandXhoIwasinsertedintoeukaryoticexpressvectorpcDNA3.1(+)togenerateanrecombinantexpressionplasmidpcDNA3.1/Du
5、IL-18(pDuIL-18),andtransformedintoEscherichiacoliJM109.Therecombinantcolonieswereidentifiedbyrestrictionenzymedigestion,PCRandsequencing.DNAsequenceconfirmedthecorrectsequenceoftherecombinanteukaryoticexpressionplasmidpDuIL-18inthereadingframeandtheligationpart.AfterthetransfectionofpDuIL-18int
6、oCos7cells,duckIL-18mRNAwasexpressedinCos7cel1.TheSDS-PAGEanalysisshowedthattheexpressedduckIL-18proteinhadmolecularweightof23000D.Theresultsofmethylthiazolyltetrazolium(MTT)assayshowedthatduckIL-18proteinexpressedinCos7cellcouldinducesignificantlytransformationofduckTlymphocytes.Immunoenhancem
7、enteffectofrecombinantexpressionplasmidpDuIL18onavianinfluenzavaccinewasobservedbyproliferationresponseoftheTlymphocytesfromspleen.Itcanobviouslyenhancethecell-mediatedimmuneresponse.Keywords:interleukin-18,duck,eukaryoticexpressi
