鸡干扰素刺激基因12(ISG12)的克隆表达及抗病毒活性分析

Cloning,expression and antiviral activity of chicken ISG12

干扰素刺激基因12(interferon-stimulated gene 12,ISG12)在病毒感染过程中已经被监测到,但是针对其抗病毒活性的研究却很少。本研究的主要目的是体外表达鸡的ISG12基因,以禽流感病毒(avian influenza viru,AIV)1215株为病毒模型,利用细胞培养检测其抗病毒活性。利用RT-PCR方法扩增鸡胚成纤维细胞(CEF)的ISG12基因,经测序分析,该基因序列与GenBank中登录的禽属ISG12基因序列相似性为100%。将该基因亚克隆至pET-32a载体构建重组原核表达质粒p32a-C12;工程菌p32a-C12/BL21经IPTG诱导表达,重组蛋白经Ni 2+琼脂糖凝胶柱层析纯化,获得相对分子质量约30 000的目的蛋白。利用ISG12蛋白预处理CEF和鸡外周血淋巴细胞(peripheral blood lymphocyte,PBL)后感染AIV 1215株,或者将该病毒与ISG12蛋白一起接种CEF细胞和PBL细胞,AIV在CEF中的复制能力与对照组无明显差异,而ISG12蛋白预处理鸡PBL细胞和同时感染AIV 1215株时的病毒含量均比对照低。体外抗病毒活性试验表明,ISG12蛋白能够抑制AIV 1215株在PBL细胞中的复制,但在CEF细胞上的作用不明显。

ISG12has been detected in viral infections,but its antiviral activity is not elucidated yet.Primary objective of this study was to express chicken-derived ISG12 and tested its antiviral activity in cell culture using AIV 1215 stain as a model virus system.cDNA gene encoding ISG12 from CEF cells was amplified by RT-PCR and sequenced.Following sequence confirmation showing100% homology to those reported in GenBank,we cloned the amplified fragment into pET-32 aand obtained a recombinant plasmid,designated p32a-C12.After induction in BL21 bacteria by IPTG,we detected an expressed product with 30 000 in size through SDS-PAGE.The soluble ISG12 protein was further purified using Ni-chelate affinity chromatography and subsequently subjected to antiviral activity experiments involving AIV 1215 strain.We conducted the AIV infection in pretreated CEF cells and PBL cells with ISG12,and the inoculation of AIV into CEF and PBL cells with ISG12 together.There were no differences in AIV replication from the control by real-timequantitative analysis in CEF cells.However,the virus titers in PBL cells were lower significantly than control.These data suggested that ISG12 protein may inhibit AIV replication in PBL cells but has no distinct antiviral activity against AIV in CEF cells.