Chinese Journal of Tissue Engineering Research ›› 2011, Vol. 15 ›› Issue (46): 8671-8675.doi: 10.3969/j.issn.1673-8225.2011.46.029

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Construction of human glucose regulated protein 78 eukaryotic expression vector and expression in retinal pigment epithelium cells

Li Man-li1, Qi Hui2, Sun Lei3, Wu Ya-zhen2   

  1. 1Henan Province Institute of Ophthalmology, Zhengzhou  450003, Henan Province, China
    2Department of Ophthalmology, Second Hospital of Jilin University, Changchun  130041, Jilin Province, China
    3Department of Ophthalmology, Fourth Affiliated Hospital of Harbin Medical University, Harbin  150000, Heilongjiang Province, China
  • Received:2011-07-29 Revised:2011-08-31 Online:2011-11-12 Published:2011-11-12
  • Contact: Wu Ya-zhen, Professor, Department of Ophthalmology, Second Hospital of Jilin University, Changchun 130041, Jilin Province, China
  • About author:Li Man-li☆, Doctor, Attending physician, Henan Province Institute of Ophthalmology, Zhengzhou 450003, Henan Province, China limanli1980@sina.com

Abstract:

BACKGROUND: Glucose regulated protein 78 (grp78) is a molecular chaperone protein, which is mostly located on the endoplasmic reticulum, and protects cells from cell apoptosis.
OBJECTIVE: To construct the pEGFP-grp78 fusion protein eukaryotic expression vector and to detect their expression in human retinal pigment epithelium (RPE) cell lines.
METHODS: The encoding fragment of human grp78 gene was obtained from a recombinant plasmid POTB7-grp78 using PCR, and it was digested by restriction enzymes Hind Ⅲ and KpnⅠ. Then the purified grp78 gene fragment was inserted into the GFP expression vector PEGFP-N1, and the recombinant plasmid PEGFP-grp78 was identified by PCR analysis and DNA sequencing. The recombinant plasmids were transfected into RPE cells by lipofectamin. RPE cell lines with grp78 stable expression were established by G418 selection. The expression of grp78 gene was detected by RT-PCR. The expression of fusion proteins was assayed by fluorescence microscopy.
RESULTS AND CONCLUSION: We successfully constructed the expression vector of pEGFP-grp78 fusion protein and the sequence results were matched to the cDNA of human grp78. Stably transfected RPE cell line was established and grp78 was expressed successfully.

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