期刊文献+

重组犬α干扰素在昆虫细胞中的高效表达与抗病毒活性鉴定

High-efficiency expression and antiviral activity of recombinant canine interferon-αexpressed in insect cells
分享 导出
摘要 目的利用杆状病毒表达系统制备犬α干扰素(Canine interferon-α,CaIFN-α)。方法根据昆虫细胞密码子偏爱性优化CaIFN-α基因,构建含双拷贝CaIFN-α优化基因的载体pFastBacDual-CaIFNα-CaIFNα,转化DH10Bac感受态后获得重组基因组rBacmid-CaIFNα-CaIFNα,转染Sf9细胞获得重组杆状病毒rBac-CaIFNα-CaIFNα,接种Sf9细胞表达rCaIFN-α。采用PCR和透射电子显微镜鉴定重组杆状病毒的外源基因与病毒形态,采用间接免疫荧光染色法检测rCaIFN-α蛋白的表达,采用细胞病变抑制法测定rCaIFN-α抑制水疱性口炎病毒(VSV-GFP)在MDCK细胞的复制能力。结果重组杆状病毒rBac-CaIFNα-CaIFNα构建成功,rCaIFN-α在rBac-CaIFNα-CaIFNα感染的Sf9细胞中表达,其抗病毒活性为8.73×105 IU/ml。结论构建的杆状病毒表达系统能高效表达rCaIFN-α,该干扰素具有高效抗病毒活性,这为犬干扰素制剂的研究奠定了基础。 Objective To efficiently express canine interferon-αusing a baculovirus/insect cell expression system.Methods The canine interferon-αgene(CaIFN-α)was optimized based on preferred codons in insect cells.The vector pFastBacDual-CaIFNα-CaIFNαcontaining the 2 copies of the CaIFN-αgene was transformed into DH10 Bac host bacteria to obtain the recombinant plasmid rBacmid-CaIFNα-CaIFNα.The recombinant baculovirus rBac-CaIFNα-CaIFNαwas obtained by transfecting Sf9 cells,and rCaIFN-αwas expressed by inoculation of Sf9 cells.The recombinant baculovirus was identified using PCR and transmission electron microscopy.CaIFN-αprotein was identified using IFA.Cytopathic inhibition was used to determine the ability of CaIFN-αprotein to inhibit the replication of vesicular stomatitis virus(VSVGFP)in MDCK cells. Results The rBacmid-CaIFNα-CaIFNαplasmid was verified as correct using restriction endonuclease analysis and DNA sequencing.The rBac-CaIFNα-CaIFNαbacmid was verified as correct using PCR and transmission electron microscopy.In IFA,specific green fluorescence was observed in Sf9 cells infected with rBac-CaIFNα-CaIFNα,indicating the expression of interferon.An antiviral activity assay indicated that recombinant CaIFN-αefficiently inhibits the replication of VSV-GFP in MDCK cells.The level of biological activity of recombinant CaIFN-αwas as high as 8.73×105 IU/ml. Conclusion This study successfully developed Sf9 cells expressing CaIFN-αwith a high level of antiviral activity.This finding lays the foundation for the further development and utilization of canine interferon.
作者 燕丽娜 傅倩芸 孙培录 赵忠欣 王威 盖微微 郑学星 郑文文 夏咸柱 YAN Li-na;FU Qian-yun;SUN Pei-lu;ZHAO Zhong-xin;WANG Wei;GAI Wei-wei;ZHENG Xue-xing;ZHENG Wen-wen;XIA Xian-zhu(College of Public Health,Shandong University,Jinan,China,250012;nstitute of Materia Medica,Shandong Academy of Medical Sciences;Military Veterinary Institute,Academy of Military Medical Sciences)
出处 《中国病原生物学杂志》 CSCD 北大核心 2019年第1期12-16,22共6页 Journal of Pathogen Biology
基金 山东省自然科学基金项目(No.ZR2016CQ18) 中国博士后科学基金项目(No.2015M580592,2016T90637) 中央高校基本科研业务费专项资金资助项目(No.2018JC050).
关键词 犬干扰素α 杆状病毒表达系统 昆虫细胞 抗病毒活性 CaIFN-α baculovirus expression system insect cells antiviral activity
作者简介 通讯作者:郑文文,E-mail:zhengwenwen@sdu.edu.cn;通讯作者:郑学星,E-mail:zhengxx2513@163.com;燕丽娜(1994-),女,山西人,硕士研究生,主要从事分子病毒学研究。E-mail:linay2012@163.com
  • 相关文献

参考文献6

二级参考文献48

  • 1王艳,王海震,曹瑞兵,陈溥言.犬α干扰素基因的高效表达及其活性测定[J].中国病毒学,2005,20(2):189-192. 被引量:20
  • 2葛丽 ,李震 ,于瑞嵩 ,刘惠莉 ,张德福 ,周智爱 ,尹逊和 .猪α干扰素基因在毕赤酵母中的分泌表达[J].中国兽医学报,2005,25(3):289-292. 被引量:27
  • 3韩春来,吴志光,史喜菊,张灿,柏亚铎,汪明.惠阳胡须鸡Ⅱ型干扰素基因在毕赤酵母中的表达及其产物活性分析[J].中国兽医科学,2006,36(4):311-314. 被引量:4
  • 4Gurkan C, Ellar DJ. Recombinant production of bacterial toxins and their derivatives in the methylotrophic yeast Pichia pastoris [J]. Microb Cell Fact, 2005, 4 (1) : 33-40. 被引量:1
  • 5Taira O, Watanugi I, Hagiwara Y, et al. Cloning and expression of canine interferon-alpha genes in Escherichia coli [J]. J Vet Med Sci, 2005, 67 (10): 1059-1062. 被引量:1
  • 6Clare JJ, Rayment FB, Ballantine SP, et al. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene [J]. Biotechnology(NY), 1991, 9 (5): 455-460. 被引量:1
  • 7Clare JJ, Romanos MA, Rayment FB, et al. Production of mouse epidermal growth factor in yeast:high-level secretion using Pichiapastoris strains containing multiple gene copies [J]. Gene, 1991, 105 (2): 205-212. 被引量:1
  • 8Sreekrishna K, Brankamp RG, Kropp KE, et al. Strategies for op- timal synthesis and secretion of heterologous proteins in the methy- lotrophic yeast Pichia pastoris [J]. Gene, 1997, 190 ( 1 ) : 55-62. 被引量:1
  • 9Li Z, Hong G, Wu Z, et al. Optimization of the expression of hepatitis B viruse gene in Pichia pastoris and Immunological characterization of the product [J]. J Biotechnol, 2008, 138 (1-2): 1-8. 被引量:1
  • 10Wang H, Wang Q, Zhang F, et al. Protein expression and purica- tion of human Zbtb7A in Piehia pastoris via gene codon optimization and synthesis [J]. Protein Expr Purif, 2008, 60 (2): 97-102. 被引量:1

共引文献23

投稿分析

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部 意见反馈