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1、四川大学学报(医学版)2007;38(4):578-582JSichuanUniv(MedSciEdi)LactoferricinB基因与几种变异克隆的构建及其在酿酒酵母中的表达与鉴定△冯永潜,查晓军,翟朝阳四川大学华西基础医学与法医学院生物化学与分子生物学教研室(成都610041)【摘要】目的构建牛乳铁多肽(LactoferricinB)的真核表达质粒pYES2/LactoferricinB,实现其在酿酒酵母S.cerevisiae中的表达,并初步检测其不同变异的体外抗菌活性。方法通过分别合成Lactoferr
2、icinB基因两条单链的部分序列,让其互为模板、引物进行PCR扩增,得到LactoferricinB与3种变异的基因序列。将它们克隆到穿梭质粒pYES2中,构建pYES2/LactoferricinB及3种变异基因重组质粒,转化到大肠杆菌Top10中,让其大量增殖。提取重组质粒经纯化后将其转化到S.cerevisiae中,通过营养缺陷型筛选获得重组酵母菌并通过半乳糖诱导使其表达目的蛋白。经离子交换柱纯化收集目的蛋白后,通过体外抑菌实验比较LactoferricinB及3种变异重组蛋白的抗菌活性。结果经PCR扩增检
3、验、DNA测序表明成功构建pYES2/LactoferricinB与3种变异基因重组质粒。提取诱导后的蛋白进行SDS-PAGE电泳及质谱检测,证实目的蛋白的存在,相对分子质量约为3.4×103。在大肠埃希菌及金黄色葡萄球菌的抑菌实验中观测到LactoferricinB和A17-LactoferricinB产生了抑菌圈。结论成功构建pYES2/LactoferricinB及变异基因重组质粒,该重组质粒能在S.cerevisiae中诱导表达目的蛋白,为进一步研究其生物功能及抗菌活性奠定了基础。【关键词】抗菌肽乳铁蛋白
4、B基因序列变异酵母表达【中图分类号】Q78Clone,Construct,ExpressionandVerificationofLactoferricinBGeneandSeveralSequenceMutationsinYeast△.DepartmentofBiochemistryandS.cerevisiaeFENGYong-qian,ZHAXiao-jun,ZHAIChao-yangMolecularBiology,WestChinaSchoolofPreclinicialandForensicMedici
5、ne,SichuanUniversity,Chengdu610041,China【Abstract】ObjectiveToconstructtheeucaryoticrecombinantplasmidofpYES2/LactoferricinBexpressinginyeastofS.cerevisiae,ofwhichtheexpressedproteinantibacterialactivitywasverifiedinpreliminary.MethodsByself-templatePCRmethod,
6、thegeneofLactoferricinBanditsseveralsequencemutationswereamplifiedwiththepartsofthepre-synthesizedsinglechains.AndthenLactoferricinBgeneanditsmutantswereclonedintothevectorofpYES2toconstructtherecombinedexpressionplasmidpYES2/LactoferricinBetc.extractedanduse
7、dtotransformtheyeastS.cerevisiae.Theexpressionsofproteinsweredeterminedafterinducedbygalactose.Theexpressionproteinswerecollectedandpurifiedbyhydronium-exchangecolumn,andthebacterialinhibitedtestwasappliedtoidentifytheproteinantibacterialactivities.ResultsThe
8、PCRamplifyingandDNAsequencingtestsindicatedthatthepurposeplasmidcontainedtheLactoferricinBgeneandseveralmutations.TheinducedtargetproteinswereconfirmedbySDS-PAGEelectrophoresisandmassspec
