重组狮头鹅α干扰素的制备及其抗病毒活性  

Preparation and antiviral effect in vitro of recombinant interferon-alpha of Shitou goose

作　　者：郑晓灵[1,2] 刘艳芬[1] 陈绍红[3] 刘铀[3] 

机构地区：[1]广东海洋大学农学院,广东湛江524088 [2]湛江市第二十中学,广东湛江524005 [3]广东海洋大学生化中心,广东湛江524088

出　　处：《中国兽医科学》2010年第7期733-737,共5页Chinese Veterinary Science

摘　　要：用PCR方法扩增了狮头鹅α干扰素(IFN-α)成熟肽编码序列,并克隆至pET-32a(+)表达载体,将重组质粒转入E.coliBL21(DE3)感受态细胞并用IPTG诱导表达。SDS-PAGE、Western-blot检测结果表明,重组狮头鹅IFN-α融合蛋白(rGoIFN-α)的分子质量大小约36ku,能与IFN-α抗血清发生特异性结合反应。rGoIFN-α包涵体经过变性、超滤纯化和复性,得到可溶性rGoIFN-α。rGoIFN-α经肠激酶酶切(eGoIFN-α)、亲和纯化获得狮头鹅α干扰素蛋白(GoIFN-α)。rGoIFN-α经Western-blot检测证实是GoIFN-α蛋白。采用微量细胞病变抑制法分析α干扰素的抗病毒活性,复性后纯化的rGoIFN-α的抗病毒活性达1.42×104U/mg;eGoIFN-α的抗病毒活性达8.23×104U/mg,GoIFN-α抗病毒活性达3.34×105U/mg。结果表明,rGoIFN-α能够抑制新城疫病毒Roakin株在鸡胚成纤维细胞中复制。The interferon-α gene of Shitou goose was amplified from genome DNA,the segment coding mature peptide of the GoIFN-α gene was cloned into prokaryotic expression vector pET32a（＋）to construct recombinant plasmid.The rGoIFN-α fusion protein was expressed under IPTG induction.Using SDS-PAGE,a protein band with molecular weight of 36ku was observed.The inclusion body were purified by degeneration,ultrafiltrate purification and renaturation.The rGoIFN-α fusion protein was digested by enterokinase to cleave the fusion peptide,which could be combined to Ni-NTA affinity resin.The effluent containing GoIFN-α was collected and condensed.The rGoIFN-α was detected by Western-blot,and only single band was observed.The results showed that the purified rGoIFN-α could specifically react with interferon antibody.The microneutralization test was used to evaluate the antiviral activity of rGoIFN-α.When the chicken embryo fibroblast cell（CEF）was treated with Newcastle disease virus（NDV）of 50 TCID_ 50,the antiviral titration of rGoIFN-α was 1.42×10 4 U/mg and the antiviral titration after digestion by enterokinase was 8.23×10 4 U/mg,while,the antiviral titration of purified cleaved GoIFN-α fusion peptide was 3.34×10 5 U/mg.The results showed that the rGoIFN-α could inhibit NDV replication in CEF.

关 键 词：狮头鹅 Α干扰素 新城疫病毒 抗病毒活性 

分 类 号：S852.42[农业科学—基础兽医学]