Preparation of recombinant PTD-HSP27 and verification of its ability to penetrate the cell membrane of human lens epithelial cells and rabbit cor-nea
10.3969/j.issn.1000-4718.2015.01.026
- VernacularTitle: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
- Author:
Lian LIU
;
Rongjie YU
;
Yun DAI
;
Zhixing ZENG
;
Xiaoling GUO
;
Qingshan JI
;
Jingxiang ZHONG
- Publication Type:Journal Article
- Keywords:
Protein transduction domain;
Heat shock protein 27;
Trans-activators of transcription;
Lens epi-thelial cells
- From:
Chinese Journal of Pathophysiology
2015;(1):135-140
- CountryChina
- Language:Chinese
-
Abstract:
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 .
