ASP-ChIFN-α融合基因的构建及表达  

Construction and Expression of ASP-ChIFN-α Fusion Gene

作　　者：李丽[1] 周学章[1] 李敏[1] 王玉炯[1] 

机构地区：[1]宁夏大学西部特色生物资源保护与利用教育部重点实验室,宁夏银川750021

出　　处：《宁夏大学学报（自然科学版）》2008年第2期157-160,共4页Journal of Ningxia University(Natural Science Edition)

基　　金：国家自然科学基金资助项目(30560110)

摘　　要：利用PCR技术从宁夏地方三黄肉鸡肝组织中扩增出α干扰素基因,构建成原核重组表达质粒pET-28a/ChIFN-α.从埃希氏菌属大肠杆菌(Escherichia coli)中扩增出L-天(门)冬酰胺酶Ⅱ(L-Asparaginase,ASP)信号肽基因,并与原核重组质粒pET-28a/ChIFN-α连接,构建成分泌型表达重组质粒pET-28a/ASP-ChIFN-α.将重组质粒经IPTG诱导5 h,用SDS-PAGE分析表达蛋白含量,并用鸡α-干扰素ELISA(Western-blot)试剂盒检测表达的重组蛋白的特异性.结果表明,包涵体型重组质粒pET-28a/ChIFN-α和分泌型重组质粒pET-28a/ASP-ChIFN-α在23 kD处均表达出目的蛋白,蛋白含量分别为27%和38%,pET-28a/ASP-ChIFN-α的蛋白表达含量比pET-28a/ChIFN-α的蛋白表达含量要高出11%,表达的重组蛋白具有特异性.实验实现了鸡α-干扰素成熟蛋白基因的高效表达,筛选出了可应用于临床的鸡α-干扰素高效表达菌株BL21(DE3)(pET-28a/ASP-ChIFN-α).To construct the recombinant plasmid pET-28a/ChIFN-a which would express inclusion complex of the exogenous protein, the chicken＇s interferon-a gene was cloned by PCR technique from the liver of Sanhuang chicken in Ningxia. The L-asparaginase Ⅱ signal peptide gene was cloned from Escherichia coli, then the signal peptide gene was linked on the recombinant plasmid pET-28a/ChIFN-α to construct the recombinant plasmid pET- 28a/ASP-ChIFN-α which would express the secreted exogenous protein. At last, the recombinant plasmid was induced for 5 hours by IPTG. The protein expression content was analyzed by SDS-PAGE electrophoresis. By using the chicken interferon-α ELISA or Western-blot kit, the characteristic of expression recombinant protein was detected. The results suggested that the expression protein molecular weight of pET-28a/ChIFN-α and pET-28a/ ASP-ChIFN-α both were 23 kD, the expression protein content of them were 27% and 38%. That means the expression protein content of pET-28a/ASP-ChIFN-α was higher than pET-28a/ChIFN-α＇s for eleven percents, and the recombinant protein has the specific characteristic. The efficient expression of chicken＇s interferon-α gene was achieved, and the bacterium BL21 （DE3）（pET-28a/ASP-ChIFN-α） expressing chicken＇s interferon-α gene efficiently was screened out, and can be applied to clinical.

关 键 词：鸡α-干扰素基因 L-天(门)冬酰胺酶Ⅱ信号肽 克隆 表达 

分 类 号：Q78[生物学—分子生物学]