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.
出处
《浙江农业学报》
CSCD
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)
关键词
绍兴鸭
Α-干扰素
基因克隆
原核表达
Shaoxing duck
interferon-α
gene cloning
prokaryotic expression
