类弹性蛋白多肽融合犬β干扰素的表达纯化和抗病毒活性及体外半衰期的研究

Purification, antiviral activity and in vitro half-life of elastin-like polypeptide-fused canine interferon-β

为了研制长效犬β干扰素(Ca IFNb)基因工程产品,用聚合酶链反应从犬基因组DNA扩增Ca IFNb成熟肽编码序列,插入类弹性蛋白多肽(ELP)融合表达载体,将重组载体转化大肠杆菌BLR(DE3),用IPTG诱导Ca IFNb-ELP表达,利用ELP介导的温度敏感可逆相变循环(ITC)进行纯化,用50%细胞病变抑制试验检测干扰素活性,用50%小鼠血浆孵育法检测体外半衰期,并与融合组氨酸标签的His-Ca IFNb进行比较。结果显示,在≤28℃条件下,90%Ca IFNb-ELP为可溶性表达,His-Ca IFNb以不溶性包涵体表达;在优化条件下进行1轮ITC,获得的Ca IFNb-ELP的纯度为96%,显著高于以包涵体纯化的His-Ca IFNb的纯度(85%);Ca IFNb-ELP的抗病毒活性为1×105 U/mg,比His-Ca IFNb的抗病毒活性高10倍;Ca IFNb-ELP的体外半衰期>24 h,显著长于His-Ca IFNb的体外半衰期(12 h)。结果表明,Ca IFNb-ELP具有表达水平高、纯化简单和体外半衰期较长等优点,有望作为长效干扰素开发利用。

To develop the genetic engineering product of long-acting canine interferon-β（Ca IFNb）,the coding sequence for the mature peptide of Ca IFNb was amplified from the genomic DNA and cloned into an elastin-like polypeptide（ELP）fusion expression vector.The recombinant vector was transformed into Escherichia coli BLR（DE3）strain and the expression of Ca IFNb-ELP fusion protein was induced with IPTG.The fusion protein was purified using ELP-mediated temperature-sensitive inverse transition cycling（ITC） and its antiviral activity was measured by 50% cytopathic effect inhibition assay.The in vitro half-life of Ca IFNb-ELP was determined by plasma stability assay using His-Ca IFNb as the control.The result showed that about 90% of Ca IFNb-ELP was expressed as a soluble protein at≤28 ℃,whereas His-Ca IFNb was expressed as inclusion bodies under all temperatures tested.After once cycle of ITC under the optimized conditions,the purity of Ca IFNb-ELP was up to 96%,which was significantly higher than that（85%）of His-Ca IFNb purified as inclusion bodies.Ca IFNb-ELP had an antiviral activity of 1×105U/mg,which was 10-fold higher than that（1×104 U/mg） of His-Ca IFNb.The in vitro half-life of Ca IFNb-ELP was 〉24 h,which was significantly longer than that（12 h） of His-Ca IFNb.These data suggestthat the Ca IFNb-ELP had the advantages of high expression level,simple purification and longer half-life with the potential for further development as a long-acting interferon.