人GPC3基因真核表达载体的构建及重组蛋白表达纯化  

Construction of eukaryotic plasmid of human GPC3 and expression and purification of recombinant GPC3 protein

作　　者：宋海晶[1] 张卓梅[2] 邱伟成[3] 杨小盼[3] 万德友[3] 何椿鹏[3] 李萌萌[3] 高新[1,3] 

机构地区：[1]解放军第306医院急诊部,北京100850 [2]武警总医院妇产科,北京100039 [3]军事医学科学院放射与辐射医学研究所药理毒理研究室,北京100850

出　　处：《细胞与分子免疫学杂志》2012年第9期937-939,943,共4页Chinese Journal of Cellular and Molecular Immunology

摘　　要：目的:人磷脂酰肌醇蛋白3是肝癌的诊断标志物和潜在治疗靶点。本研究目的在于获得足量的GPC3蛋白用于结构、功能及应用研究。方法:通过RT-PCR从人胎肝中克隆GPC3编码cDNA,利用Xho I和Xba I酶切位点,将GPC3ORF编码基因插入毕赤酵母表达载体pPICZ A中,构建真核表达质粒pPICZ A-GPC3。以该表达质粒转染毕赤酵母菌株GS115,在含有Zeocin的YPD平板上进行筛选。然后使用HRP标记的山羊抗人IgG在醋酸-硝酸纤维素双层膜上进行Dot blot筛选。对筛选获得的菌株进行诱导表达,表达上清经SDS-PAGE和Western blot检测。结果:筛选获得的阳性菌株诱导表达上清SDS-PAGE结果表明在预期位置观察到GPC3重组蛋白条带,且该蛋白条带与抗GPC3单克隆抗体(mAb)孵育后呈现特异性反应。工程菌株经高密度培养后,发酵上清中重组GPC3表达量约5 mg/L,进而通过阳离子交换层析从发酵上清中纯化了重组蛋白。结论:利用毕赤酵母表达并纯化了重组人GPC3蛋白,为进一步研究GPC3的结构和功能奠定了基础。AIM：To obtain enough human glypican-3（GPC3） protein for structural and functional research.METHODS： The full-length cDNA coding for GPC3 was cloned by RT-PCR from human fetal hepatocytes.The open reading frame（ORF） of the cDNA consists of 1 700 bases,encoding a mature protein of 556 amino acids.The cDNA was inserted into the pPICZ A vector to construct a expression plasmid,named pPICZ A-GPC3.Then the plasmid was transformed into a Pichia pastoris strain,GS115 and the positive strains were screened on the YPD plates with Zeocin.The positive strains were further screened on cellulose acetate and nitrocellulose membrane with HRP labeled His-tag antibody.The selected strains were induced by methanol and the supernatants were analyzed by SDS-PAGE and Western blotting.RESULTS： SDS-PAGE analysis showed an anticipated band on the gel that could bind with goatanti-GPC3 antibody.Furthermore,the strain was fermented and the expression level was about 5 mg/L,and the recombinant GPC3 protein was purified by cation-exchange chromatography from the fermentation supernatant.CONCLUSION： Human GPC3 was expressed successfully in Pichia pastoris and purified to obtain the recombinant protein from fermentation supernatant,which made it possible for further structural and functional studies on GPC3.

关 键 词：人磷脂酰肌醇蛋白3 克隆 毕赤酵母 表达 纯化 肝癌 

分 类 号：Q816[生物学—生物工程]