猪β干扰素成熟蛋白编码基因的克隆及其在大肠埃希菌中的表达与纯化  

Cloning, Expression and Purification of Porcine Interferon-β Mature Protein in Escherichia coli

作　　者：吴阳[1] 李玲玲[2] 李玉林 李晨阳[1] 王岁楼[1] 

机构地区：[1]郑州轻工业学院食品与生物工程学院,河南郑州450002 [2]河南师范大学生命科学学院,河南新乡453007 [3]河南省生物工程技术研究中心,河南郑州450002

出　　处：《动物医学进展》2006年第12期75-78,共4页Progress In Veterinary Medicine

摘　　要：从猪肝脏中提取基因组DNA,利用PCR法直接扩增了猪β干扰素成熟蛋白编码基因。将PCR产物亚克隆至pGEM-7Zf(+)载体,转化入大肠埃希菌TG1,筛选出阳性菌株后进行测序。序列分析结果表明,克隆片段含编码猪β干扰素成熟蛋白质的495个核苷酸,与GenBank上(登录号:S41178)的序列同源性达到100%。在此基础上,构建了猪β干扰素原核表达载体pQE30/poIFN-β并转化入大肠埃希菌M15。该工程菌在37℃经0.03mmol/LIPTG诱导4h后,进行SDS-PAGE电泳,结果在分子质量约20ku的位置出现了明显的目的蛋白条带,表达产物主要以包涵体形式存在。经凝胶分析软件Bandscan分析,表达产物约占菌体总蛋白的18%。包涵体用6mol/L盐酸胍溶解,经过Ni-NTA亲和层析法纯化,获得了纯度较高的目的蛋白,为下一步对猪β干扰素进行复性及活性研究打下了基础。Porcine genome DNA was isolated from porcine liver cell, and the gene coding porcine interferon-β（poIFN-β） mature protein was directly amplified by PCR. The gene was sub-cloned into pGEM-7Zf（＋） vector, then transformed into E. coli TG1. The sequencing result suggested that the gene coding poIFN-β mature protein was composed of 495 bp, which had the identities of 100 % with the polFN-β mature protein gene published in the GenBank （Accession No. S41178）. Then, the gene was inserted into pQE30 vector and was transformed into E. coli M15. After induced at 37 ℃ for 4 hours by adding IPTG to a final concentration of 0. 03 mmol/L, a specific expression band with a relative molecular weight 20 ku was detected by SDS-PAGE,and the expression product existed mainly in inclusion body which accounted for about 18% of total cell protein. The inclusion body was dissolved in 6 mol/L guanidine chloride and subsequently purified by Ni-NTA affinity chromatography. Highly purified poIFN-β was obtained. These studies provided a basic work in research of poIFN-β＇s renaturation and biological activity.

关 键 词：猪β干扰素成熟蛋白 基因克隆 原核表达 亲和纯化 

分 类 号：S852.4[农业科学—基础兽医学] Q785[农业科学—兽医学]