MDCK细胞ISG15基因的克隆表达及抗病毒活性分析

浙江农业学报

MDCK细胞ISG15基因的克隆表达及抗病毒活性分析

（1 山东师范大学生命科学学院，山东济南 250014；2山东省农业科学院家禽研究所，山东济南 250023；3 山东农业大学动物科技学院，山东泰安 271018）

出版日期:2015-02-25 发布日期:2015-03-12

Clone and expression of ISG15 gene from MDCK cell and analysis of its antiviral activity

（1 College of Life Sciences， Shandong Normal University， Jinan 250014， China;2 Institute of Poultry Science， Shandong Academy of Agricultural Sciences， Jinan 250023， China;3 College of Animal Science and Technology， Shandong Agricultural University， Taian 271018， China）

Online:2015-02-25 Published:2015-03-12

摘要/Abstract

摘要： 为了解 ISG15基因体外抗病毒机制，应用RT\|PCR方法扩增了MDCK细胞的ISG15基因，经测序分析，该基因序列与GenBank中登录的犬属ISG15基因序列相似性为99%。将该基因亚克隆至pET\|28a载体构建出重组原核表达质粒p28a\|M15；工程菌p28a\|M15/BL21经IPTG诱导表达，获得了约22 kD的重组目的蛋白，重组蛋白经Ni2+柱层析纯化。于H9N2亚型禽流感病毒S2株感染前后在MDCK细胞中定量加入纯化的ISG15蛋白，结果显示，病毒感染MDCK细胞前加入ISG15蛋白可以抑制病毒的复制，但病毒感染后添加该蛋白则促进了病毒的复制。

关键词: ISG15, MDCK细胞系, 抗病毒活性, H9N2

Abstract: ISG15 gene was cloned from MDCK cells using RT\|PCR， and the alignment of ISG15 with the reported sequence （GenPept databank: XM_0036390532） revealed 99 % nucleotide sequence identities. The ISG15 was cloned into expression vector pET\|28a to construct the expression plasmid p28a\|M15， and was expressed in Escherichia coli. The protein expression in E. coli induced by IPTG was secreted in the supernatant with a recombinant protein of about 22 kD. The recombinant protein was purified using Ni\|chelate affinity chromatography and then added into MDCK cells quantitatively with and without AIV H9N2 subtype infection. The results showed that ISG15 protein could inhibit AIV replication in normal MDCK cells， however， it could promote AIV replication in MDCK cells infected by AIV H9N2 subtype. This study will provide theoretical basis for ISG15 antiviral function in vitro.

Key words: ISG15, MDCK cell line, antiviral activity, H9N2

刘爱玲1，潘杰1，庞运倩3，于可响2，马秀丽2，宋敏训2，*. MDCK细胞ISG15基因的克隆表达及抗病毒活性分析[J]. 浙江农业学报.

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