绍兴鸭α-干扰素基因克隆、表达及活性测定  

Cloning,expression, and activity determination of Shaoxing duck IFN-α gene

作　　者：叶伟成[1] 张存[1] 王一成[1] 刘蔓雯[1] 徐丽华[1] 袁秀芳[1] 张燕[1] 

机构地区：[1]浙江省农业科学院畜牧兽医研究所,浙江杭州310021

出　　处：《浙江农业学报》2005年第3期115-119,共5页Acta Agriculturae Zhejiangensis

基　　金：浙江省重点农业科技项目(2005C22034)

摘　　要：采用PCR法从绍兴鸭的肝组织基因组DNA中扩增出α干扰素基因,插入pUC18载体,进行序列测定及分析;将成熟α干扰素基因亚克隆到表达载体pET 28α(+)中,并转化入大肠杆菌BL21进行表达;表达产物经复性后,进行抗病毒活性的测定。测序结果表明,该基因(sxDuIFN α)由576个核苷酸组成,共编码191个氨基酸。该基因与GenBank公布的鸭α干扰素基因(X84764,AB128861)的核苷酸序列同源性分别为99.3%和99.1%,氨基酸序列同源性均为97.9%。表达载体经IPTG诱导表达出相对分子量为20.7kD的DuIFN α。该产物经细胞病变抑制法测定,显示出具有明显的抗猪水泡性口炎病毒(VSV)及鸭瘟弱毒疫苗毒的活性。<Abstrcat>Using PCR method,Shaoxing duck interferon-alpha gene (SX-DuIFN-α) was amplified from the genomic DNA of Shaoxing duck liver and cloned into vector pUC18. After sequencing, the gene of mature SX-DuIFN-α protein was sub-cloned into expression vector pET-28α (+) and expressed in E.coli BL21-plysS. After renaturation, the antiviral activity of the expression product was determined. The results of sequencing showed that sxDuIFN-α consisted of 576 nucleotides, encoding 191 amino acids. The homologies of the sxDuIFN-α, DuIFN-α in GenBank and BDIFN-α nucleotide sequences were 99.3% and 99.1%, respectively, and amino acid homology was 97.9% among them. By induction with IPTG, the expression vector pET-28α (+) containing sxDuIFN-α gene had expressed DuIFN-α with relative molecular weight of 20.7 kD.This expression product was verified to be of high antiviral activity against VSV and attenuated duck plague virus by inhibiting the cytopatic effect.

关 键 词：绍兴鸭 Α-干扰素 基因克隆 原核表达 

分 类 号：S852.4[农业科学—基础兽医学]