p75NTR-Fc融合蛋白在毕赤酵母中的表达、鉴定和活性分析

Expression, Identification and Biological Activity of Recombinant Human p75NTR-Fc in Pichia pastoris

为在酵母细胞中表达出具有生物活性的p75NTR Fc融合蛋白 ,采用PCR方法分别扩增α因子 (α factor)和p75NTR Fc基因 ,经重叠PCR ,获得α factor p75NTR Fc基因 .DNA序列分析该融合基因后 ,插入pAO815载体并构建串联多拷贝表达载体pAO815 3α factor p75NTR Fc .重组质粒电转化酵母GS115细胞后摇瓶培养 ,1%甲醇诱导表达的融合蛋白经ProteinA亲和层析和SephadexG 10 0纯化 ,Western印迹、N末端测序进行蛋白质鉴定 ,ELISA及细胞培养进行生物活性分析 .SDS PAGE分析显示 ,表达产物以可溶性分子形式存在于培养上清中 ,诱导第 4d的表达量最高 ,占上清总蛋白 6 0 %以上 ,ProteinA纯化后有 2条蛋白带 ,免疫印迹分析这 2条蛋白带均能和抗p75及抗IgG抗体结合 ,N末端序列测定证实 1条为完整p75NTR Fc ,1条为其蛋白酶降解带 .ELISA等分析表明 ,p75NTR Fc能与NGF结合 ,p75NTR Fc能抑制NGF对PC12细胞的分化作用 .

p75NTR-Fc with biological activity was expressed, purified and identified for the further study of its pharmacology application. The gene of α-factor and p75NTR-Fc was amplified by PCR, and then the fused full length gene of α-factor-p75NTR-Fc was obtained by overlap PCR, and was inserted into a Pichia pastoris vector pAO815 to construct a recombinant expression plasmid pAO815-3α-factor-p75NTR-Fc after getting a tandem of multiple copies of expression cassettes. The recombinant plasmid was transformed into Pichia pastoris cell line GS115 by electroporation. The transformants were screened, cultured and induced by the addition of 1% methanol. The expressed fusion protein was purified by Protein A affinity chromatography and Sephadex G-100, and identified by SDS-PAGE and N-terminal amino acids sequence analysis. The biological activity was analyzed by Western blotting, ELISA and cell culture. The expression of p75NTR-Fc reached up to 60% of the total proteins in medium supernatant as shown by SDS-PAGE. There were two proteins after purified by Protein A and both were binding with anti p75NTR and rabbit anti-IgG antibody. Amino acids sequence analysis showed that one of the proteins was integrity and another was the degradation product. p75NTR-Fc could interact with NGF and inhibit NGF effect on PC12 cell differentiation.