Chinese Journal of Tissue Engineering Research ›› 2010, Vol. 14 ›› Issue (50): 9361-.doi: 10.3969/j.issn.1673-8225.2010.50.013

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Effects of human BIGH3 cloning and expression on keratocytes adhesion and migration

Ge Hong-yan1, Shi Yan1, Yang Fan1, Zhang Yi1, Liu Ping1, Liu Han-ruo2   

  1. 1 Ophthalmology Hospital, the First Hospital of Harbin Medical University,  Harbin  150001, Heilongjiang Province, China; 2 University of East Anglia, Norwich NR4 England
  • Online:2010-12-10 Published:2010-12-10
  • Contact: 刘平，主任医师，哈尔滨医科大学附属第一医院眼科医院，黑龙江省哈尔滨市 150001 Ping_Liu53@hotmail.com
  • About author:Ge Hong-yan☆, Doctor, Attending physician, Ophthalmology Hospital, the First Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China ge.hongyan@hotmail.com
  • Supported by:

    the Tackle Key Program in Heilongjiang Province, No. GC08C416*; the Foundation of the First Hospital of Harbin Medical University, No. B08-004*; the Education Department of Heilongjiang Province, No. 11551267*

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Abstract:

BACKGROUND: BIGH3 protein locates at corneal epithelium and substantia propria layer. Previous studies have found that BIGH3 protein can promote wound healing of corneal epithelium, thus, we further explore the effects of BIGH3 protein on wound healing.
OBJECTIVE: To construct a prokaryotic expression plasmid encongding BIGH3 gene, and to investigate its effects on rabbit keratocytes and extracellular matrix adhesion and migration.
METHODS: The ORF of BIGH3 was PCR-amplified, digested by KpnI and SalI, and ligated into pET32a(+). Then, the reconstructed plasmid was identified with PCR, enzyme digestion and sequencing. The plasmid was transformed into E. coli BL21(DE3), and induced to express fusion protein with IPTG and purified with Ni-NTA-His affinity chromatography. The expression of BIGH3 was detected by SDS-PAGE and Weston Blot. The effects of recombinant Pet32a/bigh3 on adhesion of cultured rabbit keratocytes were assayed by MTT. Keratocytes migration assays were performed in transwellplates.
RESULTS AND CONCLUSION: PCR amplification, double digestion and DNA sequence demonstrated that recombinant plasmid have shown that the size of the inserted pET32a(+) fragment was as expected. The fusion protein formed inclusion body with IPTG and was 78 kD using SDS-PAGE. Meanwhile, the expressed product showed a good binding ability to anti-BIGH3 monoclonal antibody by Weston blot. MTT assay displayed BIGH3 promoted the adhesion of rabbit kertocytes. BIGH3 protein increased keratocytes migration. We successfully clone and express BIGH3 gene and purify recombinant BIGH3 protein, and verified the bioactivity through recombinant BIGH3 protein promoting cells adhesion and migration.

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