資源描述：
《豬α1 干擾素基因的克隆與原核表達(dá)》由會(huì)員上傳分享，免費(fèi)在線(xiàn)閱讀，更多相關(guān)內(nèi)容在教育資源-天天文庫(kù)。
1、中國(guó)農(nóng)學(xué)通報(bào)2010,26(11):1-6ChineseAgriculturalScienceBulletin豬α1干擾素基因的克隆與原核表達(dá)1211楊永勝，陳春花，宋勤葉，左玉柱（1河北農(nóng)業(yè)大學(xué)動(dòng)物科技學(xué)院，河北保定071001；2河北省邯鄲市動(dòng)物疫病預(yù)防控制中心，河北邯鄲056005）摘要：根據(jù)豬α1干擾素（pocineinterferon-alpha1，poIFN-α1）的全基因序列（EU364896）設(shè)計(jì)合成1對(duì)特異引物，通過(guò)RT-PCR從三元雜交仔豬外周血淋巴細(xì)胞擴(kuò)增出poIFN-α1全基因，將該基因克隆到p
2、GEM-T載體，構(gòu)建重組質(zhì)粒pGEM-T-poIFN-α1，進(jìn)行測(cè)序。以pGEM-T-poIFN-α1為模板，經(jīng)PCR擴(kuò)增帶有酶切位點(diǎn)的poIFN-α1基因，將其定向克隆到原核表達(dá)載體pGEX-6P-1中，構(gòu)建重組表達(dá)載體pGEX-6P-poIFNα1，轉(zhuǎn)化大腸桿菌BL21（DE3）進(jìn)行蛋白誘導(dǎo)表達(dá)和鑒定，并確定蛋白最佳表達(dá)條件。序列分析表明，克隆的poIFN-α1基因全長(zhǎng)546bp，編碼181個(gè)氨基酸，前23個(gè)氨基酸組成信號(hào)肽，無(wú)糖基化位點(diǎn)。SDS-PAGE和Westernblotting證實(shí)重組表達(dá)菌經(jīng)IPTG誘導(dǎo)
3、后，可表達(dá)相對(duì)分子質(zhì)量約46ku的融合蛋白（GST-poIFN-α1）。當(dāng)以1.0mmol/L的IPTG于37℃下誘導(dǎo)表達(dá)9h時(shí)，蛋白表達(dá)量最高。poIFN-α1基因的克隆與表達(dá)為進(jìn)一步研究其抗病毒活性，開(kāi)發(fā)病毒治療和疫苗免疫增強(qiáng)用生物制劑奠定了基礎(chǔ)。關(guān)鍵詞：豬α1干擾素；克??；原核表達(dá)；鑒定中圖分類(lèi)號(hào)：S852.4文獻(xiàn)標(biāo)志碼：A論文編號(hào)：2009-2772CloneandProkaryoticExpressionofPorcineInterferon-α1Gene1211YangYongsheng,ChenChunh
4、ua,SongQinye,ZuoYuzhu(1CollegeofAnimalScienceandTechnology,HebeiAgriculturalUniversity,Baodinghebei071001;2HandanCenterforAnimalDiseaseControlandPrevention,Handanhebei056005)Abstract:Apairofspecificprimerforporcineinterferon-alpha1(poIFN-α1)wasdesignedandsynthes
5、izedaccordingtogenesequence(EU364896),andpoIFN-α1genewasamplifiedbyRT-PCRfromperipheralsbloodlymphocytes(PBLC)ofternaryhybridpiglet.ThenpoIFN-α1completegenewasclonedintopGEM-Tvectorandsequenced.Moreover,poIFN-α1genewithenzymesiteswasclonedintoprokaryoticexpressi
6、onvectorpGEX-6P-1toconstructrecombinantexpressionplasmidpGEX-6P-poIFN-α1andtransformitintoE.coliBL21(DE3).RecombinantpoIFNα1wasexpressedandidentifiedwithexpressionconditionsconfirmed.TheanalysisofsequenceindicatedthattheclonedpoIFN-α1geneconsistedof546bpencoding
7、181aminoacidswithasignalpeptideof23aminoacidsbutwithoutglycosylationsite.Theselectedrecombinantexpressedapproximatelya46kufusionproteinwithglutathioneS-transferase(GST)andpoIFN-α1whichwasdemonstratedbySDS-PAGEandWesternblotting.Theamountoffusionproteinreachedthe
8、highestwhentherecombinantswereinducedfor9hourson37℃with1.0mmol/LIPTG.CloneandexpressionofpoIFN-α1genelaidafoundationforthestudyofantiviralactivityandutilizationofpoIF
