重组鸭γ-干扰素的表达与抗病毒活性分析

Expression and antiviral activity of recombinant duck γ-interferon

为表达重组鸭γ-干扰素(DuIFNγ),并鉴定其在体内、体外抗病毒活性,本研究根据大肠杆菌密码子的偏好性对GenBank中登录的DuIFNγ序列进行优化并合成,将其克隆至p ET30a-ELP表达载体中进行重组蛋白诱导表达,并利用类弹性蛋白多肽(ELP)的可逆相变循环特性进行纯化,结果显示,重组蛋白(rELP-DuIFNγ)正确表达,纯化后的rELP-DuIFNγ纯度达90%以上。通过细胞病变抑制法,在VSV/MDCK系统和VSV/DEF系统中检测rELP-DuIFNγ体外抗病毒活性,结果显示,rELP-DuIFNγ在VSV/MDCK系统和VSV/DEF系统中的抗病毒活性分别为为4.17×104U/mg和4.54×105U/mg。利用雏鸭感染DHAV-1,同时灌服rELP-DuIFNγ,观察并计算雏鸭发病时间与存活率,初步研究rELP-DuIFNγ的体内抗病毒作用,结果显示r ELP-DuIFNγ在体内可以延缓雏鸭发病时间、降低其发病率。以上结果表明,rELP-DuIFNγ在大肠杆菌中可高效表达,在体内外均具有一定的抗病毒活性。本研究为基因工程DuIFNγ制剂的研制及临床应用奠定了基础。

In order to analyze the expression and antiviral activity of recombinant duck interferon-γ(DuIFNγ) in vivo and in vitro,the whole sequence encoded DuIFNγpublished in GenBank was synthesiezd after the codon optimization according to the condon bias of E.coli.And the synthetic sequence was cloned into pET30a-ELP,vector and expressed in E.coli.The resulting recombinant protein was purified by using the reversible phase change cycling characteristics of ELP Elastin-like peptide.As a result,the DuIFNγwas expressed well,and the purity of the purified recombinant protein was over 90%.The analysis of antiviral activity was performed in vitro in ducklings by using the cytopathy inhibition method in VSV/MDCK system and VSV/DEF system.The antiviral activities were 4.17×10~4U/mg and 4.54×10~5U/mg,respectively.DHAV-1 and ELP-DuIFNγwere simultaneously inoculated in the ducklings,and the onset time and survival rate of the ducklings were observed and calculated The results showed that both the pathogenesis time and the disease incidence of ducklings were delayed and reduced with the treatment of DuIFNγin vivo.Taking together,that recombinant DuIFNγcan be efficiently expressed in E.coli and had antiviral activity in vitro.This study can lay the foundation for the development and clinical application of genetic engineering DuIFNγpreparations in the futrue.