云芝免疫调节蛋白基因克隆及酵母重组表达

Gene Cloning and Yeast Recombinant Expression of Fungal Immunomodulatory Protein from Coriolus versicolor

真菌免疫调节蛋白(Fip)是真菌中重要的药理成分之一。为进一步开发真菌中Fip资源,从云芝中克隆了云芝免疫调节蛋白基因Fip-cve,对Fip-cve的理化性质和生物信息学进行了研究,并将其转入酵母表达载体中,成功获得了酵母转化子。结果表明:Fip-cve基因扩增片段长为336 bp,该序列编码111个氨基酸;Fip-cve蛋白的二级结构是以无规卷曲为主,同时含有35.71%的延伸链,还含有12.35%的α-螺旋,主要位于N端;疏水性分析表明该基因编码的蛋白是亲水性蛋白,二级结构分析也解释了该蛋白的结构特征;SDSPAGE电泳结果显示Fip-cve目的蛋白均在酵母中获得了高效表达,目的蛋白分子量约为13 ku;生物信息学分析结果表明,Fip-cve与灵芝属真菌免疫调节蛋白具有很高的同源性,特别是与小孢灵芝具有更高的同源性。该研究初步建立了Fip-cve的真核表达体系,以期为Fip-cve的进一步开发提供参考。

Fungal immunomodulatory protein （Fip） is one of the major pharmacological components of fungi. To exploit FIPs in fungi, we cloned Fip-cve gene from Coriolus versicolor, analyzed bioin- formatics of Fip-eve, and constructed yeast expression vector with Fip-cve. Especially, we success- fully obtained yeast transformants for the first time. The results indicated that length of the amplified fragment of Fip-cve gene was 336 bp, which was encoded by 111 amino acids, and the secondary structure of Fip-cve prote was mainly based on random coil, while containing 35.71% extended strand and 12. 35% a-helix, mainly located in the N-terminus. Hydrophobieity analysis also sugges- ted that protein encoded by this gene was a hydrophilie protein, while the secondary structure analy- sis explained the structural features of the protein. SDS-PAGE electrophoresis showed that Fip-cve target protein was highly expressed in yeast and molecular weight of the protein was about 13 ku. Bioinformatics analysis showed Fip-cve had high homology with the immunomodulatory protein from species of Gcmoderma, and especially with that of the smaller spore species, G.lucidum. The eukary- otic expression system we established for Fip-cve has laid a solid foundation for potential biotechno-logical exploitation of this protein.