牛γ干扰素基因的原核表达及其单克隆抗体的研制
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- 英文论文题名：Expression of Bov IFN-γ and Development of Monoclonal Antibodies against Bov IFN-γ
- 论文作者：钱琨 ; 赵巍 ; 黄大卢 ; 邵红霞 ; 秦爱建
- 英文论文作者：QIAN Kun ; ZHAO Wei ; HUNAG Dalu ; SHAO Hongxia ; QIN Aijian ; Key Laboratory of Jiangsu Preventive Veterinary Medicine ; Yangzhou University ; Ministry of Education Key Lab for Avian Preventive Medicine ; Yangzhou University ; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses ; Yangzhou University
- 年：2016
- 作者机构：扬州大学江苏省动物预防医学重点实验室;扬州大学教育部禽类预防医学重点实验室;扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心;
- 论文关键词：牛γ干扰素 ; 原核表达 ; 单克隆抗体
- 英文论文关键词：BoIFN-γ ; prokaryotic expression ; monoclonal antibody
- 会议召开时间：2016-07-20
- 会议录名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集
- 语种：中文
- 分类号：S852.4
- 学会代码：SYGW
- 会议名称：中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会
- 会议地点：中国内蒙古通辽
- 主办单位：中国畜牧兽医学会兽医公共卫生学分会
- 学会名称：中国畜牧兽医学会兽医公共卫生学分会
- 页数：9
- 文件大小：724k
- 原文格式：D
- 会议级别：全国
摘要
为了制备高质量高效价抗牛γ干扰素(Bo IFN-γ)的单克隆抗体,本研究将Bo IFN-γ基因克隆到His标签的大肠杆菌表达载体p ET32a+中,诱导表达并纯化蛋白作为抗原免疫Balb/C小鼠制备单克隆抗体。结果筛选到9株单抗,分别命名为Bo IFN-γ-1E10、Bo IFN-γ-3C2、Bo IFN-γ-3E1、Bo IFN-γ-6C6、Bo IFN-γ-6H9、Bo IFN-γ-6B6、Bo IFN-γ-6E5、Bo IFN-γ-2A10、Bo IFN-γ-2G10。ELISA、Western blot免疫蛋白印迹和间接免疫荧光实验结果显示,制备的单克隆抗体与原核、真核表达的Bo IFN-γ都具有良好的反应性。本研究制备获得的针对牛γ干扰素的单克隆抗体为建立检测天然Bo IFN-γ的方法提供了重要的生物材料。
To generate monoclonal antibodiesagainst Bo IFN-γ,the fragment of Bo IFN-γ in the p GEX-6P-1 vector was sbucloned into p ET32 a vector.The purified His-Bo IFN-γfusion protein was injected into Balb/C mice as immunogen.Nine positive monoclonal antibodies,named as Bov IFN-γ-1E10、Bov IFN-γ-3C2、Bov IFN-γ-3E1、Bov IFN-γ-6C6、Bov IFN-γ-6H9、Bov IFN-γ-6B6、Bov IFN-γ-6E5、Bov IFN-γ-2A10 and Bov IFN-γ-2G10,were obtained.The results of ELISA,Western blot and IFA demonstrated that these monoclonal antibodies are able to recognize not only prokaryotic but also eukaryotic expression Bo IFN-γ protein.The generated monoclonal antibodies in this study will provide important reagents for further establishment of a sandwich ELISA for the detection of natural Bo IFN-γ.
To generate monoclonal antibodiesagainst Bo IFN-γ,the fragment of Bo IFN-γ in the p GEX-6P-1 vector was sbucloned into p ET32 a vector.The purified His-Bo IFN-γfusion protein was injected into Balb/C mice as immunogen.Nine positive monoclonal antibodies,named as Bov IFN-γ-1E10、Bov IFN-γ-3C2、Bov IFN-γ-3E1、Bov IFN-γ-6C6、Bov IFN-γ-6H9、Bov IFN-γ-6B6、Bov IFN-γ-6E5、Bov IFN-γ-2A10 and Bov IFN-γ-2G10,were obtained.The results of ELISA,Western blot and IFA demonstrated that these monoclonal antibodies are able to recognize not only prokaryotic but also eukaryotic expression Bo IFN-γ protein.The generated monoclonal antibodies in this study will provide important reagents for further establishment of a sandwich ELISA for the detection of natural Bo IFN-γ.
引文
[1]Borden EC,Sen GC,Uze G,et al.Interferons at age 50:past,current and future impact on biomedicine[J].Nat Rev Drug Discov,2007 Dec;6(12):975-90.
[2]J.A.Langer,Interferon at 50:New molecules,new potential,new(and old)questions[J].Sci.STKE 2007,2007pe53.
[3]Bonjardim CA,Ferreira PC,Kroon EG.Interferons:signaling,antiviral and viral evasion[J].Immunol Lett,2009 Jan 29;122(1):1-11.
[4]李彤,张以芳,罗佳琴,牛γ-干扰素在牛结核检测中的研究应用[J].中国畜牧兽医,2006.33(11):p.53-56.
[5]Daniluk,J.and M.Kandefer-Szerszen,Interferon production by peripheral blood cells of patients with alcoholic liver disease(ALD)[J].Arch Immunol Ther Exp(Warsz),1994.42(3):p.231-8.
[6]Kandefer-Szerszen,M.,et al.,Suppression of interferon response of bovine leukocytes during clinical and subclinical ketosis in lactating cows[J].Dtsch Tierarztl Wochenschr,1992.99(11):p.440-3.
[7]Desem,N.and S.L.Jones.Development of a human gamma interferon enzyme immunoassay and comparison with tuberculin skin testing for detection of Mycobacterium tuberculosis infection[J].Clin Diagn Lab Immunol,1998.5(4):p.531-6.
[8]Ben Selma,W.Interferon gamma+874T/A polymorphism is associated with susceptibility to active pulmonary tuberculosis development in Tunisian patients[J].DNA Cell Biol,2011.30(6):p.379-87.
[9]Wood,P.R.,L.A.Corner,and P.Plackett,Development of a simple,rapid in vitro cellular assay for bovine tuberculosis based on the production of gamma interferon[J].Res Vet Sci,1990.49(1):p.46-9.
[10]Gonzalez Llamazares,O.R.,et al.,Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain[J].Vet Microbiol,1999.70(1-2):p.55-66.
[11]许金俊,奶牛干扰素-γ基因的表达及其在乳房炎防治中的初步应用[D].扬州:扬州大学博士学位论文,2005.
[12]许金俊,秦.,刘岳龙等,奶牛干扰素基因克隆及其在大肠杆菌中表达[J].扬州大学学报,2003.24(1):5-9.
[13]尹天燕.γ-干扰素介导牛结核快速检测方法的研究及初步应用[D].扬州:扬州大学硕士学位论文,2010.
[14]Kashima,T.,et al.,Expression of bovine cytokines in Escherichia coli[J].J Vet Med Sci,1999.61(2):p.171-3.
[15]Krupnova,O.F.,N.I.Sizova,and A.G.Smolianitskii,[Increase in the yield of recombinant proteins in Saccharomyces cerevisiae yeasts as a result of optimizing their culture conditions][J].Prikl Biokhim Mikrobiol,1995.31(3):p.311-5.
[16]Wedlock,D.N.,et al.,Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils[J].J Dairy Res,2000.67(2):p.189-97.
[17]Rothel,J.S.,et al.,The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle:conditions affecting the production of gamma-interferon in whole blood culture[J].Aust Vet J,1992.69(1):p.1-4.Raggo,C.,et al.,The in vivo effects of recombinant bovine herpesvirus-1 expressing bovine interferon-gamma[J].J Gen Virol,2000.81(Pt 11):p.2665-73.
[2]J.A.Langer,Interferon at 50:New molecules,new potential,new(and old)questions[J].Sci.STKE 2007,2007pe53.
[3]Bonjardim CA,Ferreira PC,Kroon EG.Interferons:signaling,antiviral and viral evasion[J].Immunol Lett,2009 Jan 29;122(1):1-11.
[4]李彤,张以芳,罗佳琴,牛γ-干扰素在牛结核检测中的研究应用[J].中国畜牧兽医,2006.33(11):p.53-56.
[5]Daniluk,J.and M.Kandefer-Szerszen,Interferon production by peripheral blood cells of patients with alcoholic liver disease(ALD)[J].Arch Immunol Ther Exp(Warsz),1994.42(3):p.231-8.
[6]Kandefer-Szerszen,M.,et al.,Suppression of interferon response of bovine leukocytes during clinical and subclinical ketosis in lactating cows[J].Dtsch Tierarztl Wochenschr,1992.99(11):p.440-3.
[7]Desem,N.and S.L.Jones.Development of a human gamma interferon enzyme immunoassay and comparison with tuberculin skin testing for detection of Mycobacterium tuberculosis infection[J].Clin Diagn Lab Immunol,1998.5(4):p.531-6.
[8]Ben Selma,W.Interferon gamma+874T/A polymorphism is associated with susceptibility to active pulmonary tuberculosis development in Tunisian patients[J].DNA Cell Biol,2011.30(6):p.379-87.
[9]Wood,P.R.,L.A.Corner,and P.Plackett,Development of a simple,rapid in vitro cellular assay for bovine tuberculosis based on the production of gamma interferon[J].Res Vet Sci,1990.49(1):p.46-9.
[10]Gonzalez Llamazares,O.R.,et al.,Field evaluation of the single intradermal cervical tuberculin test and the interferon-gamma assay for detection and eradication of bovine tuberculosis in Spain[J].Vet Microbiol,1999.70(1-2):p.55-66.
[11]许金俊,奶牛干扰素-γ基因的表达及其在乳房炎防治中的初步应用[D].扬州:扬州大学博士学位论文,2005.
[12]许金俊,秦.,刘岳龙等,奶牛干扰素基因克隆及其在大肠杆菌中表达[J].扬州大学学报,2003.24(1):5-9.
[13]尹天燕.γ-干扰素介导牛结核快速检测方法的研究及初步应用[D].扬州:扬州大学硕士学位论文,2010.
[14]Kashima,T.,et al.,Expression of bovine cytokines in Escherichia coli[J].J Vet Med Sci,1999.61(2):p.171-3.
[15]Krupnova,O.F.,N.I.Sizova,and A.G.Smolianitskii,[Increase in the yield of recombinant proteins in Saccharomyces cerevisiae yeasts as a result of optimizing their culture conditions][J].Prikl Biokhim Mikrobiol,1995.31(3):p.311-5.
[16]Wedlock,D.N.,et al.,Effects of yeast expressed recombinant interleukin-2 and interferon-gamma on physiological changes in bovine mammary glands and on bactericidal activity of neutrophils[J].J Dairy Res,2000.67(2):p.189-97.
[17]Rothel,J.S.,et al.,The gamma-interferon assay for diagnosis of bovine tuberculosis in cattle:conditions affecting the production of gamma-interferon in whole blood culture[J].Aust Vet J,1992.69(1):p.1-4.Raggo,C.,et al.,The in vivo effects of recombinant bovine herpesvirus-1 expressing bovine interferon-gamma[J].J Gen Virol,2000.81(Pt 11):p.2665-73.