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1、第21卷第3期南京医科大学学报2001年5月ACTAUNIVERSITATISMEDICINALISNANJING175利用Bac-to-Bac表达系统高效表达胰岛素样生长因子Ⅱ122赵红,汪承亚,段宇,P.H.Steenbergh,J.S.Sussenbach,陈家伟12(南京医科大学第一附属医院内分泌科,血液科,江苏南京210029;LaboratoryforPhysiologicalChemistry,UtrechtUniversi-ty,TheNetherlands)[摘要]目的:利用Bac-to-Bac表达系统高效表达
2、成熟的胰岛素样生长因子Ⅱ(IGF-Ⅱ)。方法:首先构建含IGF-ⅡcDNA的重组供体质粒pFastBac1,然后重组pFastBac1所含的转位元件mini-Tn7,在感受态细胞DH10Bac内转位到粘粒上的连接位点mini-attTn7构成重组粘粒,重组粘粒转染昆虫细胞Sf9产生重组杆状病毒,重组杆状病毒进行感染Sf9细胞。结果:琼脂糖凝胶分析显示重组供体质粒pFastBac1的成功构建;琼脂糖凝胶分析重组粘粒的PCR产物表明已获得重组粘粒;十二烷基磺酸钠(SDS)-PAGE和WesternBlotting显示7KD左右蛋白质
3、条带的出现。结论:利用Bac-to-Bac表达系统成功表达具有免疫学活性的IGF-Ⅱ,可用于临床疾病的诊治。[关键词]重组人胰岛素样生长因子Ⅱ;Bac-to-Bac表达系统;昆虫细胞[中图分类号]R394.3;Q786[文献标识码]A[文章编号]1007-4368(2001)03-0175-04HighExpressionofInsulin-likeGrowthFactor-Ⅱ(IGF-Ⅱ)UsingBac-to-BacExpre-ssionSystem122ZHAOHong,WANGCheng-ya,DUANYu,P.H.St
4、eenbergh,J.S.Sussenbach,CHENJia-wei1(DepartmentofEndocrinology,DepartmentofHemotology,theFirstAffiliatedHospitalofNan-2jingMedicalUniversity,JiangsuNanjing210029,China;LaboratoryforPhysiologicalChemistry,UtrechtUniversity,TheNetherlands)[Abstract]Objective:Inordertoo
5、btainmatureinsulin-likegrowthfactor-Ⅱ(IGF-Ⅱ),weusedBac-to-Bacbaculovirusexpressionsystem.Methods:FirstlytheIGF-ⅡcDNAwasclonedintoadonorplasmidpFastBac1andtherecombinantpFastBac1wasthenintroducedintocompetentcellsDH10Bac.Recombinantbacmidswerecon-structedbytransposing
6、amini-Tn7elementfromadonorplasmidpFastBac1tothemini-attTn7attachmentsiteonthebacmidwheretheTn7transpositionfunctionswereprovidedintransbyahelperplasmid,andthenusedtotransfectSf9insectcellstogetrecombinantbaculovirus.Therecombinantbaculoviruswasusedtoinfectinsectcells
7、.Results:AgarosegelanalysisshowedthatrecombinantdonorplasmidpFastBac1wasconstructedsuccessful-ly;AgarosegelanalysisofPCRproductsconfirmedrecombinantbacmid;SDS-PAGEandWesternBlottingshowedthata7KDproteinbandappeared.Conclusion:ThematureIGF-Ⅱwithimmunogenecityhasbeenex
8、-pressedandproducedbyusingBac-to-Bacexpressionsystem.[Keywords]insulin-likegrowthfactor-Ⅱ;Bac-to-Bacexpressionsystem;insectcells[Ac
