抗nestin多克隆抗体的制备.pdf

《抗nestin多克隆抗体的制备.pdf》由会员上传分享，免费在线阅读，更多相关内容在应用文档-天天文库。

1、第四军医大学学报(JFourthMilMedUniv)2005;26(8)http://ournal.fmmu.edu.cn691研究原著文章编号I1000-2790(2005)08-0691-03抗nestin多克隆抗体的制备李军林1!韩骅1!兰莉2!杨曦1!李军锋1!王亚蓉3!祝仰庭1(第四军医大学:1基础部医学遗传学与发育生物学教研室9陕西西安71003392唐都医院儿科93唐都医院放射科9陕西西安710038)三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三三

2、三三三三PreparationofpolyclonalantibodytoWesternBlotting和免疫组化方法分析其特异性.结果I6>~is-nestin-C融合蛋白免疫新西兰兔成功获得多克隆抗nestin体9WesternBlotting和免疫组化结果显示制备的抗体具有较19~AN~ua19LANLi29YANGXi19LIJun-LIJun-Lin高的特异性.结论I构建了pET-32a-nestin-C表达载体9并在19WANGYa-Rong39Z~UYang-Ting1Feng大肠杆菌中获得高效表

3、达.该多克隆抗体效价高~特异性好.1DepartmentofMedicalGeneticsDevelopmentBiology9[关键词)nestin基因"融合表达"抗体制备SchoolofBasicMedicine9FourthMilitaryMedicalUniversity9[中图号)@785[文献标识码)A23Xian7100339DepartmentofPediatrics9DepartmentofRadi-ology9Tangdu~ospital9FourthMilitaryMedicalUnive

4、rsity9Xian7100389China0引言巢蛋白(nestin)是中间丝蛋白9在胚胎发育早期9[Abstract)AIM:TocloneandexPresstheC-fra9mentofcDNa[1]在神经上皮干细胞和发育中的骨骼肌中强表达.在encodin9mousenestinPePtideandtoobtainsPecificPolyclonal一些目前仍缺乏有效标记的干细胞研究领域也发挥着antibody.METHODS:TheC-fra9mentofcDNaencodin9mouse重要作用9

5、如角膜缘干细胞[2]等.在大鼠胰腺发育的胚nestinPePtideWasobtainedWiththemethodofRT-PCR.Thecor-胎后期~新生期~成年三阶段9胰岛内nestin阳性细胞rectlyidentifiedseCuenceWasclonedintotheProkaryoticex-[3]数呈下降趋势.我们选取同源性相对较高的片段进PressionvectorPET-32atoconstructPET-32a-nestin-CexPres-行反转录PCR和原核表达等方法9期望获得特异性

6、巢sionvector.6>His-nestin-CfusionProteinWasexPressedinE.蛋白抗体用于相关研究.coliBL21WithlPTginduction.afterPurification9thefusionPro-teinWasusedtoimmunizeNeWZealandrabbitsto9ettherabbitanti-nestinserum.ThesPecificityofthePloyclonalanti-nestinan-l材料和方法tibodyWasanalyzed

7、byWesternBlottin9andimmunohistochemi-l.l材料反转录试剂盒为Promega产品.E14胚calmethods.RESULTS:ThePloyclonalanti-nestinantibodyWas胎cDNA文库~大肠杆菌XL109BL21为本室保存;质successfullyobtainedbyimmunizin9NeWZealandrabbitsWith6>粒pET-32a~各种限制性内切酶~pMD18-T载体~rTaIHis-nestin-CfusionProtein.

8、ThesPecificityofantibodyWasconfirmedDNA聚合酶~SolDNA连接酶~异丙基硫化-l-D-半byWesternBlottin9andimmunohistochemicalmethods.CONCLU-乳糖苷(PTG)为Takara产品;质粒提取盒~胶回收试SION:PET-32a-nestin-CexPressionvectorisconstruct