生精相关基因mTSARG3的原核表达和抗体制备.pdfVIP

窑2070 窑 南方医科大学学报(JSouthMedUniv) 2010;30(9) 生精相关基因mTSARG3 的原核表达和抗体制备 刘 刚袁卢光琇渊中南大学生殖与干细胞工程研究所袁湖南长沙410078冤 摘要院 目的 构建小鼠生精相关基因pGEX-KG/mTSARG3 重组载体袁 进行原核表达和多克隆抗体制备遥 方法应用 RT-PCR 从小鼠睾丸 cDNA 文库中扩增mTSARG3 的开放阅读框袁T-A 克隆后将mTSARG3 插入到原核表达载体 pGEX-KG 中袁 经测序鉴定后将重组质粒转入宿主菌E.coliBL21袁IPTG 诱导表达GST/mTSARG3 融合蛋白袁 采用 SDS和Westernblotting对融合蛋白进行分析和鉴定遥 以融合蛋白为免疫原免疫家兔袁制备抗mTSARG3 多克隆 抗体并进行Westernblotting鉴定遥 结 成功构建了pGEX-KG/mTSARG3 重组质粒袁测序结果与预期一致曰转化重组 质粒的E.coliBL21 经IPTG37 益诱导4h后高效表达GST/mTSARG3融合蛋白曰Westernblotting 实验结果显示制备抗 体可与原核表达融合蛋白特异性结合遥 结论 成功进行了mTSARG3基因的原核表达和多克隆抗体制备袁为下一步的 功能研究奠定基础遥 关键词院mTSARG3曰基因克隆曰蛋白表达曰多克隆抗体曰精子发生 中图分类号院R34;R339.21 文献标识码院A 文章编号院1673-4254渊2010冤09-2070-04 ConstructionofpGEX-KG/mTSARG3recombinantvectorandpreparationofanti-mTSARG3 polyclonalantibody LIUGang,LUGuang-xiu InstituteofReproductionandStemCellEngineering,CentralSouthUniversity,Changsha410078,China Abstract: ObjectiveToconstructpGEX-KG/mTSARG3recombinantvectorandpreparethefusionproteintoobtainrabbit polyclonalantibodyagainstmTSARG3. MethodsTheopenreadingframe (ORF) ofmTSARG3genewasamplifiedfrom mousetestiscDNAlibrarybyPCR. TheproductswereclonedintopGEM-TEasyvectorsandsequenced. Therecombinant plasmidsweredigestedby EcoRIandSalI and subcloned intoPGEX-KG vector. After identificationby DNA sequence analysis, therecombinantplasmidsweretransformedintocomponent E.coli BL21cells, andtheGST/mTSARG3fusion proteinwasexpressedwithIPTGinduction. The anti-mTSARG3 polyclonalantibodywas produced byimmunizationof rabbitswiththefusionprotein. The resultantantibodywaspurifiedbyantigen columnandusedfor Westernblottingfor detecting mTSARG3 expression. Results The recombinant vector pGEX-KG/mTSARG3 was successfully constructed. GST/mTSARG3 fusion protein was expressed abundantly at 4 h after IPTG induction and polyclonal antibodies were obtained. Westernblotanalysisd