人BIGH3基因克隆表达对兔角膜基质细胞黏附和迁移的影响

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

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KpnI和SalI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Westernblot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,Westernblot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。

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.