重组七鳃鳗phb2蛋白的原核表达及纯化

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1、重组七鳃鳗PHB2蛋白的原核表达及纯化#王颖，高杨，李铁松，李庆伟**(辽宁师范大学生命科学学院，大连，116081)510摘要：以课题组前期构建的pMD18-T-Lm-PHB2质粒为模板，PCR扩增Lm-PHB2基因全长CDS区，PCR产物经HindⅢ和EcoRI双酶切后连接至表达载体pET-32a，构建重组质粒pET-32a-Lm-PHB2。将鉴定正确的重组质粒转化入大肠杆菌RosettaBlue，经IPTG诱导表达后，表达产物通过SDS-PAGE和Westernblotting进行检测，利用镍柱亲和层析纯化得到纯度较高的目的蛋白rLm-PHB2。结果表明，经

2、PCR、双酶切和测序鉴定，所构建的重组质粒pET-32a-Lm-PHB2序列正确，表达产物于裂解菌液的上清和沉淀中均存在，经SDS-PAGE和Westernblotting证实在约47KD处有目的蛋白rLm-PHB2的表达条带。构建重组七鳃鳗PHB2蛋白的原核表达载体，并大量表达和纯化目的蛋白，为该蛋白后续的抗体制备及生物活性研究奠定了基础。关键词：海洋生物学；七鳃鳗；PHB2；原核表达；蛋白纯化中图分类号：R91515ProkaryoticexpressionandpurificationofrecombinedlampreyPHB2proteinWANGYing,GA

3、OYang,LITiesong,LIQingwei(LifeScienceCollegeofLiaoningNormalUniversity,Dalian,116081)Abstract:Objective:WeaimedtoconstructthesolubleprokaryoticexpressionvectorofPHB2,20253035obtainadequateamountofpurifiedrLm-PHB2proteinforfurtherstudyofitsbiologicalactivityinvivo.Methods:ThePHB2genewasam

4、plifiedfromthepMD18-T-Lm-PHB2plasmidtemplatewhichwaspreviouslyconstructed.ThePCRproductsweresubjectedtoHindⅢandEcoRIdigestionandthenlinkedtothesolubleexpressionvectorpET-32a.TheidentifiedrecombinantplasmidpET-32a-Lm-PHB2wastransformedintoRosettaBlue,andtheIPTGinducedexpressionofrLm-PHB2wa

5、sconfirmedbySDS-PAGEandWesternblottingassay.TherLm-PHB2fusionproteinwaspurifiedwithHisaffinitychromatographypurificationsystem,thenthehighlypurifiedproteinrLm-PHB2wasobtained.Results:ThecorrectconstructionofrecombinantplasmidpET-32a-Lm-PHB2wasconfirmedbyPCR,restrictionenzymedigestionandge

6、nesequencingidentification.Expressionproductswereverifiedtopresentinthesupernatantofbacterialysis,declaringthesuccessfulsolubleexpressionofrLm-PHB2.SDS-PAGEandWesternblottingresultsshowedthatthemolecularweightofrLm-PHB2proteinwasabout47KD,correspondingwithanticipantsize.Conclusion:ThepET-

7、32a-Lm-PHB2prokaryoticexpressionvectorwasconstructedcorrectly,andthesolubleexpressionofPHB2proteinwassucceeded.Ultimately,therLm-PHB2proteinwithhighpuritywasobtainedafterpurification.Keywords:marinebiology;lamprey;PHB2;prokaryoticexpression;proteinpurification基金项目：高