中国组织工程研究

中国组织工程研究 ›› 2010, Vol. 14 ›› Issue (50): 9361-.doi: 10.3969/j.issn.1673-8225.2010.50.013

• 组织构建实验造模 • 上一篇    下一篇

人BIGH3基因克隆表达对兔角膜基质细胞黏附和迁移的影响

葛红岩1，石妍1，杨帆1，张毅1，刘平1，刘含若○2   

  1. 1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；2 University ofEast Anglia,Norwich NR4 England
  • 出版日期:2010-12-10 发布日期:2010-12-10
  • 通讯作者: Liu Ping, Chief physician, Ophthalmology Hospital, First Hospital of Harbin Medical University, Harbin 150001, Heilongjiang Province, China Ping_Liu53@hotmail.com
  • 作者简介:葛红岩☆，女，1979年生，博士，主治医师，主要从事角膜创伤愈合及机制研究。 ge.hongyan@hotmail.com
  • 基金资助:

    黑龙江省攻关课题(GC08C416)，哈医大一院院基金(B08-004)，黑龙江省教育厅(11551267)。

     

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*

PDF

470

被引次数

2

摘要：

背景：BIGH3蛋白位于角膜上皮和基质层，在基质层高表达，先前研究已经发现BIGH3蛋白能促进角膜上皮创伤愈合，为此进一步探讨其对角膜基质创伤愈合的影响。

目的：构建人BIGH3基因的原核表达载体，观察其对角膜细胞与细胞外基质黏附和迁移的作用。

方法：PCR扩增BIGH3基因的ORF阅读框，并通过Kpn I和Sal I插入到原核表达载体pET32a(+)中。经PCR、酶切和序列测定方法鉴定重组质粒。将重组质粒转入BL21(DE3)表达，IPTG诱导表达人BIGH3融合蛋白, 并进行SDS-PAGE电泳分析和Western blot检测分析；以Ni-NTA树脂对蛋白纯化与复性，作用于体外培养的兔角膜细胞，用MTT法分析其对细胞与细胞外基质黏附的影响，采用改良的Boyden微孔膜双槽法观察BIGH3蛋白对角膜细胞迁移的影响。

结果与结论：重组质粒经PCR、酶切与DNA测序证实插入了pET32a(+)载体中。通过IPTG诱导，成功的表达融合蛋白在包涵体中，经SDS-PAGE电泳分析，出现了一条新生的蛋白条带，Western blot也证实了该蛋白具有与BIGH3抗体特异性的结合能力。MTT法和Boyden微孔膜双槽法分别证明所表达的BIGH3融合蛋白可促兔角膜细胞黏附和迁移。成功构建了人BIGH3重组融合蛋白表达质粒，纯化了该基因的原核表达产物，并通过重组融合蛋白BIGH3能促进兔角膜细胞与细胞外基质黏附及迁移，而验证了重组BIGH3蛋白的活性。

关键词: BIGH3基因, 角膜细胞, 黏附, 迁移, 组织构建

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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