密码子优化提高PRRSV GP5/M双基因在真核细胞中的同步表达  

Codon optimization enhances synchronous expression of recombinant PRRSV GP5 and M proteins in eukaryotic cells

作　　者：张永红[1,2] 徐建[3] 张建楼[1] 霍珊珊[1] 王利月[1] 林洪羽[3] 李秀锦[3] 仲飞[1] 

机构地区：[1]河北农业大学动物医学院基础兽医系/河北兽医生物技术工程技术研究中心,河北保定071001 [2]中国农科院兰州兽医研究所/家畜疫病病原生物学国家重点实验室,甘肃兰州730046 [3]燕山大学环境与化学工程学院生物工程系,河北秦皇岛066004

出　　处：《河北农业大学学报》2015年第4期92-97,115,共7页Journal of Hebei Agricultural University

基　　金：河北省自然科学基金(C2013204130);河北省教育厅重点科学研究项目(ZD20131056);家畜疫病病原生物学国家重点实验室课题(SKLVEB2013KFKT022)

摘　　要：为提高猪繁殖与呼吸障碍综合征病毒(PRRSV)GP5和M蛋白基因在动物细胞中同步表达水平,本试验依据动物偏爱的密码子对GP5和M蛋白基因分别进行了密码子优化,并利用本室构建的含内部核蛋白体进入位点(IRES)载体pcDNA-IRES,将密码子优化的和野生型的GP5基因和M蛋白基因分别插入到IRES的上下游,分别构建成GP5与His-标签融合和M蛋白与Myc-标签融合的密码子优化的和野生型的GP5/M双基因表达载体pcDNA-OpGP5/His-IRES-OpM/MH和pcDNA-GP5/His-IRES-M/MH,将双基因表达载体转染HEK293T细胞使其表达,通过Western blot检测,比较GP5和M蛋白在真核细胞的表达水平。结果显示,密码子优化可明显提高GP5和M在HEK293T细胞中的表达水平,本试验为GP5和M蛋白功能的研究提供了有利条件。To enhance the expression levels of porcine reproductive and respiratory syndrome virus（PRRSV）GP5and M protein genes in animal cells as a synchronous manner,we optimized GP5 and M genes based on animal main codons.The codon-optimized and wild-typeGP5 and M genes were inserted respectively into the upstream and downstream of inter ribosome entry site（IRES）in the IRES-containing plasmid pcDNA-IRES,constructed previously in our laboratory,to generate His-tag-fused GP5 and Myc-tag-fused M bicistronic expression vectors：the condon-optimized vector pcDNA-OpGP5/His-IRES-OpM/MH and the wild-type vector pcDNA-GP5/His-IRES-M/MH.The vectors were transfected into HEK293 Tcells mediated by calcium phosphate.The expressed GP5 and M proteins in the cells were detected by Western blot.Results showed that the codon optimization significantly enhanced the expression levels of GP5 and M protein in HEK293 Tcells as a synchronous manner.This study provides the advantages for further investigation on GP5 and M biological functions.

关 键 词：密码子优化 PRRSV GP5蛋白 M蛋白 表达水平 

分 类 号：Q78[生物学—分子生物学]