血红铆钉菇免疫调节蛋白基因克隆及其生物信息学分析和重组表达  

Gene Cloning, Bioinformatic Analysis and Recombinant Expression of a Novel Fungal Immunomodulatory Protein from Chroogomphis rutilus

作　　者：林景卫[1,2] 关山越[2] 钟鸣[2] 陈丽静[2] 李浩戈[2] 张丽[2] 范文丽[1] 李天来[1] 

机构地区：[1]沈阳农业大学园艺学院,沈阳110161 [2]沈阳农业大学生物科学技术学院,沈阳110161

出　　处：《沈阳农业大学学报》2015年第5期561-567,共7页Journal of Shenyang Agricultural University

基　　金：中国博士后科学基金项目(2013M541251)

摘　　要：为研究血红铆钉菇中克隆的一种新型真菌免疫调节蛋白基因以及其在毕赤酵母GS115中进行高效重组表达,采用同源克隆的方法从血红铆钉菇菌丝体基因组DNA中扩增得到一个新的真菌免疫调节蛋白基因——FIP-cru,构建FIP-cru真核表达重组载体,并在毕赤酵母GS115中进行FIP-cru重组表达。利用硫酸铵沉淀、琼脂糖镍纳米磁珠纯化目的重组FIP-cru蛋白,采用SDSPAGE和Western blot验证重组FIP-cru表达情况。利用体外血细胞凝集实验与MTT法检测r FIP-cru生物学活性。结果表明:FIPcru属于真菌免疫调节蛋白家族,包含342 bp,编码113个氨基酸,经生物信息学分析发现,FIP-cru与其他真菌免疫调节蛋白有较高的同源性。SDS-PAGE和Western blot检测表明FIP-cru在毕赤酵母GS115中正确重组表达,粗蛋白表达水平为148.5mg·L-1。纯化后的r FIP-cru可以在体外凝集小鼠和羊的血细胞而不凝集人血细胞,MTT法表明r FIP-cru可明显刺激小鼠脾淋巴细胞的增殖。FIP-cru为一种新型真菌免疫调节蛋白,毕赤酵母高效重组表达的r FIP-cru具有良好的生物学活性。A novel gene encoding a fungal immunomodulatory protein（FIP-cru） was cloned and effectively expressed yeast Pichia pastoris GS115. Homology-based cloning was used to obtain a new gene（named FIP-cru） from the genomic DNA of the edible mushroom Chroogomphis rutilus. Then FIP-cru was integrated into an eukarotic expression vector p PIC9 and transformed in P. pastoris.The recombinant FIP-cru was expressed by induction of methanol in P. pastoris GS115. r FIP-cru was purified via ammonium sulfate precipitation and agrose nickel magnetic nanobeads, and confirmed by SDS-PAGE and Western blot. The blood cell agglutination and MTT method were used to examine the activities of the r FIP-cru. The FIP-cru, a member of the fungal immunomodulatory protein family,is composed of 342 bps encoding a protein containing 113 amino acid, and is highly homologous with other FIPs. In addition, r FIP-cru was expressed in P. pastoris GS115 with a yield of 148.5 mg·L^-1. In vitro, r FIP-cru was capable of agglutinating mouse and sheep red blood cells rather than the human＇s, which also could stimulate the cell viability of murine splencytes. The novel FIP-cru gene was functionally and effectively expressed in P. pastoris, and r FIP-cru displayed good biological activities.

关 键 词：血红铆钉菇 真菌免疫调节蛋白 毕赤酵母GS115 重组蛋白 琼脂糖镍纳米磁珠 血细胞凝集 

分 类 号：Q94[生物学—植物学]