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目的利用酵母表达系统制备人LIGHT-Fc融合蛋白。方法利用基因工程方法构建含人LIGHT胞外段基因和人Ig G4 Fc基因的重组质粒p PIC9K-LIGHT-Fc,经SalⅠ线性化后电转化感受态酵母菌GS115,PCR鉴定重组转化子。将阳性重组转化子经甲醇诱导表达后,RT-PCR法检测目的基因的转录水平,SDS-PAGE及Western blot法进行表达产物的鉴定。结果表达质粒p PIC9K-LIGHT-Fc经酶切及测序鉴定,证明构建正确。10个重组子均为Mut+型阳性转化子,经甲醇诱导后可扩增出特异性目的基因片段。表达的重组LIGHT-Fc融合蛋白相对分子质量约45 000,可与鼠抗人LIGHT多克隆抗体发生特异性结合。结论人LIGHT-Fc融合蛋白在毕赤酵母中已成功获得了表达,为进一步开展其生物学功能的研究奠定了基础。
Objective To prepare human LIGHT-Fc fusion protein using yeast expression system. Methods Recombinant plasmid p PIC9K-LIGHT-Fc containing human LIGHT extracellular domain gene and human Ig G4 Fc gene was constructed by gene engineering method. After SalⅠ linearization, the competent cell was transformed into competent yeast GS115, and the recombinant transformants were identified by PCR. After the positive recombinant transformants were induced by methanol, the transcription level of the target gene was detected by RT-PCR, and the expression products were identified by SDS-PAGE and Western blot. Results The plasmid p PIC9K-LIGHT-Fc was confirmed by restriction analysis and sequencing. All 10 recombinant plasmids were Mut + positive transformants. After induced by methanol, specific target gene fragments were amplified. The expressed recombinant LIGHT-Fc fusion protein has a relative molecular mass of about 45,000 and can specifically bind to a mouse anti-human LIGHT polyclonal antibody. Conclusion The human LIGHT-Fc fusion protein has been successfully expressed in Pichia pastoris, which laid the foundation for the further study of its biological function.