鸡粒细胞巨噬细胞集落刺激因子-猪干扰素α1融合基因在大肠埃希菌中的可溶性表达及其生物学活性研究
详细信息    查看全文 | 推荐本文 |
  • 英文篇名:Soluble expression and activity analysis of chicken granulocyte macrophage-colony stimulating factor-porcine interferon alpha 1 fusion gene in Escherichia coli
  • 作者:胡涛 ; 何志远 ; 张文昌 ; 赵俊 ; 王明丽
  • 英文作者:HU Tao;HE Zhi-yuan;ZHANG Wen-chang;ZHAO Jun;WANG Ming-li;Department of Microbiology, Anhui Medical University;Anhui Jiuchuan Biotechnology Co., Ltd.;
  • 关键词:鸡粒细胞巨噬细胞集落刺激因子(ChGM-CSF) ; 猪干扰素α1(PoIFNα1) ; 融合基因 ; 可溶性表达 ; 促淋巴细胞增殖 ; 抗病毒活性
  • 英文关键词:chicken granulocyte macrophage colony-stimulating factor(ChGM-CSF);;porcine Interferon alpha 1(PoIFNα1);;fusion gene;;soluble expression;;promoting lymphocyte proliferation;;antiviral activity
  • 中文刊名:ZGXQ
  • 英文刊名:Chinese Journal of Preventive Veterinary Medicine
  • 机构:安徽医科大学微生物学教研室;安徽九川生物科技有限公司;
  • 出版日期:2019-06-15
  • 出版单位:中国预防兽医学报
  • 年:2019
  • 期:v.41
  • 基金:安徽省教育厅高校省级自然科学重大研究项目(KJ2012ZD08)
  • 语种:中文;
  • 页:ZGXQ201906005
  • 页数:5
  • CN:06
  • ISSN:23-1417/S
  • 分类号:33-37
摘要
为可溶性表达重组鸡粒细胞巨噬细胞集落刺激因子(ChGM-CSF)与猪干扰素α1 (PoIFNα1)融合蛋白,并研究其生物学活性,本研究分别提取鸡、猪肝脏细胞总RNA,采用RT-PCR方法扩增ChGM-CSF和PoIFNα1基因,经linker连接上述两种基因后将其克隆于pET-32a原核表达载体,转化E.coli BL21 (DE3)菌株进行诱导表达,SDS-PAGE和western blot检测融合蛋白表达产物。在ST细胞/VSV病毒测定系统以细胞病变抑制法滴定该融合蛋白的抗病毒活性;同时用MTT法检测其对鸡淋巴细胞增殖活性的促进作用。结果显示,PCR扩增并融合后的ChGM-CSF和PoIFNα1融合基因约为1 000 bp,构建重组表达载体后,诱导表达的rChGM-CSF-PoIFNα1融合蛋白分子量约55 ku,主要存在于破碎菌体的上清中,表达量较高。Western blot检测结果显示,该融合蛋白分别能够与ChGM-CSF多抗和PoIFNα单克隆抗体特异性结合,其抗病毒比活性约为1.1×10~6 IU/mg,并且具有促进鸡淋巴细胞增殖的活性。本研究为rChGM-CSF-PoIFNα1重组融合蛋白的研制及其相关活性的测定提供实验依据。
        To investigate the biological activities of chicken granulocyte-macrophage colony-stimulating factor(ChGM-CSF)and porcine interferon alpha 1(PoIFNα1), the recombinant plasmid with both two genes above was expressed in vivo. In this study, these two genes were amplified by RT-PCR from the total RNAs of chicken and porcine liver cells respectively before connecting with each other by a linker. And the resulting fusion gene was inserted into pET-32a prokaryotic expression vector and its recombinant plasmid was transformed into E.coli BL21(DE3) strain. The expression products were subjected to SDS-PAGE and western blot, and their antiviral activity was determined using the ST cell/VSV assay system with the method to the cytopathic effect(CPE) reduction assay. Additionally, the MTT assay was performed to detect the activity of the recombinant protein in term of promoting the proliferation of chicken lymphocytes. The results showed that the lengths of the fusion gene was about 1,000 bp with a linker involved. The fusion gene was clone and expressed sucessfully in E.coli. The molecular weight of recombinant protein was 55 ku. The expressed proteins were mainly found to be water soluble with a good expression level. The protein could bind specifically to the monoclonal antibodies against either rChGM-CSF or r PoIFNα, and its antiviral specific activity was about 1.1×10~6 IU/mg. And it has an effect on promoting lymphocyte proliferation in chicken blood. This current study will provide some experimental experiences for the expression and activity analysis of the rChGM-CSF-PoIFNα1 recombinant fusion protein.
引文
[1]Raftery N,Stevenson N J.Advances in anti-viral immune defence:revealing the importance of the IFN JAK/STAT pathway[J].Cell Mol Life Sci,2017,74(14):2525-2535.
    [2]Lefevre F,La Bonnardiere C.Molecular cloning and sequencing of a gene encoding biologically active porcine alpha-interferon[J].J Interferon Res,1986,6(4):349-360.
    [3]Chinsangaram J,Moraes M P,Koster M,et al.Novel viral disease control strategy:adenovirus expressing alpha interferon rapidly protects swine from foot-and-mouth disease[J].J Virol,2003,77(2):1621-1625.
    [4]Teijaro J R.TypeⅠinterferons in viral control and immune regulation[J].Curr Opin Virol,2016,16:31-40.
    [5]Becher B,Tugues S.GM-CSF:From growth factor to central mediator of tissue inflammation[J].Immunity,2016,45:963-973.
    [6]国家药典委员会.《中国药典》三部通则3526,重组人粒细胞巨噬细胞刺激因子生物学活性测定法[M].北京:中国医药科技出版社,2015年.
    [7]汤仁树,赵俊,王明丽.重组猪α干扰素理化性状及其体外抗病毒活性研究[J].安徽农业科学,2010,38(08):3928-3930.
    [8]国家药典委员会.《中国药典》三部通则3523,干扰素生物学活性测定法(第一法细胞病变抑制法)[M].北京:中国医药科技出版社,2015.
    [9]Balan V,Nelson D R,Sulkowski M S,et al.A PhaseⅠ/Ⅱstudy evaluating escalating doses of recombinant human albumin-interferon-alpha fusion protein in chronic hepatitis C patients who have failed previous interferon-alpha-based therapy[J].Antivir Ther,2006,11(1):35.
    [10]Wanjalla C N,Goldstein E F,Wirblich C,et al.A role for Granulocyte-Macrophage colony-stimulating factor in the regulation of CD8+T cell responses to rabies virus[J].Virology,2012,426(2):120-133.

© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号

地址:北京市海淀区学院路29号 邮编:100083

电话:办公室:(+86 10)66554848;文献借阅、咨询服务、科技查新:66554700