hLNα4LG1基因克隆、表达及生物学活性检测

Gene cloning,expression and biological function of hLNα4LG1

目的克隆并表达具有生物学活性的人层黏连蛋白α4链LG1组件(hum an lam in in alpha4 chain LG1 modu le,hLNα4LG1)蛋白。方法RT-PCR扩增hLNα4LG1的cDNA片段并将其插入pMD-18T载体进行测序。将测序片段亚克隆入原核表达载体pET-28 a并在BL21(DE3)大肠杆菌中进行表达,W estern b lot对表达蛋白进行鉴定。用N i-NTA亲和柱对目的蛋白进行纯化,并通过细胞黏附与伸展实验对其生物学活性进行检测。结果成功克隆了hLNα4LG1的cDNA片段,SDS-PAGE及W estern b lot分析显示,经IPTG诱导后BL21(DE3)/pET28 a-LG1总蛋白中出现一条分子量为26×103的新蛋白带。与对照组相比,N i-NTA亲和层析纯化的目的蛋白有明显促进A549细胞伸展与黏附的作用。结论克隆并原核表达了hLNα4LG1蛋白,目的蛋白具有促进细胞黏附与伸展的活性,为深入研究hLNα4LG1的功能奠定了基础。

Objective To clone and express human laminin alpha4 chain LG1 module（hLNα4LG1）protein holding biological activity. Methods The cDNA encoding hLNα4LG1 was amplified by RT-PCR, then inserted into pMD-18T vector and sequenced. The hLNα4LG1 cDNA fragment was subcloned into pET-28a vector and expressed in BL21 （ DE3 ） strain. The hLNα4LG1 protein was detected by Western blotting and purified by Ni-NTA column. The biological activity of target protein was detected through cell adhesion and expansion test. Results The cDNA fragment of hLNα4LG1 was cloned successfully. While BL21 （ DE3 ）/pET-28a-LG1 bacterium was induced with IPTG, a new protein band with a relative molecular weight of 26 000 was shown. Purified hLNα4LG1 protein could promote the expansion and adhesion of A549 cells obviously, as compared with the control group. Conclusion The hLNα4LG1 was cloned and expressed successfully, on which the cells can adhere and expand.