人层黏连蛋白α4链LG1-3组件的克隆及酵母表达

Expression of LG1-3 module gene of laminin α4 chain in Pichia pastoris

目的:利用巴斯德毕赤酵母表达系统获得重组的人层黏连蛋白LG1-3组件蛋白,为进一步研究LG1-3的结构和功能间的关系奠定基础。方法:利用RT-PCR从人胎盘组织总RNA中扩增LG1-3组件cDNA,并重组入pPICZαA,电转化入毕赤酵母GS115,利用甲醇诱导表达目的蛋白。结果和结论:获得了1 800 bp LG1-3组件cDNA,利用体外定点突变将SacⅠ酶切位点GAGCTC突变为GAGCGC,经SDS-PAGE和W estern印迹检测到相对分子质量为67.48×103的LG1-3组件蛋白的表达。

Objective： To express the LG1-3 module of human laminin alpha 4 chain in P. pastoris expression system for studying the function of LG1-3 module. Methods： RT-PCR were used to obtain the cDNA of LG1-3 module, which was inserted into the P. pastoris expression vector pPICZαA to give rise to pPICZ-MLG1-3. After linearization with Sac I and transforming into P. pastoris strains GS115, the LG1-3 gene was integrated into chromosome of GS115. Results and conclusion： The 1 800 bp cDNA of LG1-3 module was obtained by RT-PCR. By in vitro site mutation, Sac I endonuclease site in 960 bp of LG 1-3 was mutated from GAGCTC into GAGCGC by PCR. With PCR and phenotype screening,a P. pastoris strain GS115 integrated with LG1-3 cDNA was obtained. Induced by methanol,the recombinant 67.48 ×10^3 LG1-3 module protein was expressed and detected by SDS-PAGE and Western blot. Further study on purification and biological function of LG1-3 recombinant protein is under way.