重组蛋白PTD-HSP27的制备及其穿细胞膜和角膜组织的功能研究  

Preparation of recombinant PTD-HSP27 and verification of its ability to penetrate the cell membrane of human lens epithelial cells and rabbit cornea

作　　者：刘莲[1] 余榕捷[2] 戴云[2] 曾智星[2] 郭晓令[2] 季青山[1] 钟敬祥[1] 

机构地区：[1]暨南大学附属第一医院眼科,广东广州510632 [2]暨南大学生物工程研究所,广东广州510632

出　　处：《中国病理生理杂志》2015年第1期135-140,共6页Chinese Journal of Pathophysiology

基　　金：国家重点基础研究发展计划(973计划)(No.2011CB707501);广东省自然科学基金资助项目(No.S2013010016037)

摘　　要：目的:制备PTD-HSP27蛋白并研究其能否穿过人晶状体上皮细胞SRA01/04细胞膜及新西兰大白兔的角膜组织进入眼前房。方法:利用重叠延伸PCR法获得重组目的基因片段PTD-HSPB1-6His,构建重组质粒p KYB-PTD-HSP27-6His,原核诱导PTD-HSP27蛋白表达及纯化重组蛋白后对其进行Western blotting鉴定;用异硫氰酸荧光素(FITC)标记重组蛋白PTD-HSP27并检测其穿透SRA01/04细胞膜的能力及兔眼角膜组织的能力。结果:成功构建并纯化制备目的蛋白PTD-HSP27,在PTD-HSP27孵育后的SRA01/04细胞及结膜囊滴注PTDHSP27兔眼前房内均可检测出重组蛋白PTD-HSP27。结论:通过重叠延伸PCR法及镍柱层析纯化法可以获得较纯的重组蛋白PTD-HSP27;重组蛋白PTD-HSP27能够穿透SRA01/04细胞膜及穿越兔眼角膜进入房水。AIM： To construct the prokaryotic expression system containing protein transduction domain （PTD） with heat shock protein 27 （HSP27） in order to prepare and purify the recombinant protein , and to verify whether the recombinant protein PTD-HSP27 has the ability to penetrate the human lens epithelial cell （ HLEC） membrane and the rabbit cornea.METHODS：The plasmid pKYB-PTD-HSPB1-6His was constructed by the technique of overlap extension PCR.The plasmid was transformed and PTD-HSP27 was purified through nickel affinity chromatography column and identi-fied by Western blotting.PTD-HSP27-6His was labeled with the fluorescein isothiocyanate （FITC）.The penetrating ability of PTD-HSP27 into HLECs and rabbit cornea was tested .RESULTS：The recombinant PTD-HSP27 plasmid was success-fully cloned and effectively expressed .The correctness of the recombinant protein PTD-HSP27 was demonstrated .Fluores-cence microscopic examination showed that PTD-HSP27-FITC was internalized by HLECs .Fluorescent labeled PTD-HSP27 was then observed in the rabbit aqueous humor .CONCLUSION：The recombined gene PTD-HSPB1 was constructed by o-verlap extension PCR technique and the PTD-HSP27 fusion protein was prepared and purified by nickel affinity chromatog-raphy column.Using the technique of PTD-fusion protein, HSP27 was transduced into HLECs and passed through the cor-nea .

关 键 词：蛋白转导域 热休克蛋白27 反式转录激活因子 晶状体上皮细胞 

分 类 号：R363[医药卫生—病理学] R77[医药卫生—基础医学]