山羊γ-干扰素的生物信息学分析及其在羊肾细胞中的瞬时表达  

Bioinformatics analysis and transient expression of CapIFN-γin goat kidney cells

作　　者：许国洋 谢三磊 沈克飞[1,2] 郑华 余远迪[1,2] 牟豪 杨柳 白运川 翟少钦[1,2] XU Guoyang;XIE Sanlei;SHEN Kefei;ZHENGG Hua;YU Yuandi;MU Hao;YANG Liu;BAI Yunchuan;ZHAI Shaoqin(Chongqing Academy of Animal Sciences,Chongqing 402460,China;Chongqing Veterinary Biological Products Engineering Technology Research Center,Chongqing 402460,China;Chongqing Youyang County Animal Disease Prevention and Control Center,Youyang,Chongqing 409800,China;Southwest University,Chongqing 402460,China)

机构地区：[1]重庆市畜牧科学院,重庆402460 [2]重庆市兽用生物制品工程技术研究中心,重庆402460 [3]重庆市酉阳县动物疫病预防控制中心,重庆409800 [4]西南大学,重庆402460

出　　处：《中国兽医学报》2023年第6期1250-1256,共7页Chinese Journal of Veterinary Science

基　　金：重庆市科研机构绩效激励引导专项资助项目(cstc2021jxjl80010);重庆市自然科学基金面上资助项目(cstc2019jcyj-msxmX0512,cstc2020jcyj-msxmX0604);重庆荣昌农牧高新技术产业研发专项资助项目(cstc2019ngzx0012)。

摘　　要：利用RT-PCR方法从山羊外周血淋巴细胞中获得山羊γ-干扰素全长基因,进行基因序列分析后,将其克隆pCDNA3.1,构建重组表达质粒pCDNA3.1-CapIFN-γ,经脂质体转染至羊肾细胞中,并进行目的蛋白的SDS-PAGE和Western blot鉴定,分析CapIFN-γ重组蛋白的表达情况。利用细胞病变抑制法测定CapIFN-γ重组蛋白对羊痘病毒的抗病毒活性。结果显示,扩增的CapIFN-γ基因序列全长为501 bp,共编码166个氨基酸,其中前23位氨基酸为信号肽序列,CapIFN-γ的α-螺旋和无规则卷曲含量较高,羊肾细胞表达的目的蛋白约为25 kD,通过对CapIFN-γ的抗病毒活性检测,发现其对羊痘病毒的增殖具有一定抑制作用,其抗病毒活性为2.2×10^(3)U/mL。本研究利用羊肾细胞成功表达了CapIFN-γ重组蛋白,并对其活性进行了初步分析,为真核表达系统获取的CapIFN-γ抗病毒作用机制的深入研究和抗山羊病毒病产品研发奠定了基础。The full length of goat interferon-(CapIFN-γ)gene was obtained from goat peripheral blood lymphocytes by RT-PCR.After gene sequence analysis,the gene was inserted into pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-CapIFN-γ.The recombinant plasmid was transfected into sheep kidney cells by liposomes.SDS-PAGE and Western blot were performed to identify the target protein expression.Using the cytopathic inhibition method,the antiviral activity of CapIFN-γrecombinant protein against goat pox virus(GTPV)was determined.The results showed that the amplified CapIFN-γgene sequence was 501 bp in length and encoded a total of 166 amino acids.Among them,the first 23 amino acids were the signal peptide sequence.CapIFN-γhad a higher content ofα-helix and random coils.The size of the target protein expressed by the cells was about 25 kDa.Through the detection of the antiviral activity of CapIFN-γ,it was found that CapIFN-γhad a certain inhibitory effect on the proliferation of GTPV,and its antiviral activity was 2.2 × 10^(3) U/mL.In this study,the CapIFN-γrecombinant protein was successfully expressed by sheep kidney cells,and its activity was preliminary analyzed,which laid a foundation for the indepth study of the antiviral mechanism of CapIFN-γobtained by the eukaryotic expression system and the development of anti-goat virus disease products.

关 键 词：山羊 Γ-干扰素 生物信息学 瞬时表达 抗病毒活性 

分 类 号：S852.2[农业科学—基础兽医学]