非洲猪瘟病毒p54蛋白的表达及单抗制备
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摘要
为制备非洲猪瘟病毒p54蛋白的单克隆抗体,本研究扩增p54蛋白的胞外区编码基因,分别克隆到p ET30a和p GEX-6p-1中,构建了p54-p ET30a和p54-p GEX-6p-1重组质粒;将质粒分别转化到大肠杆菌BL21中,表达C末端带6个His、N端带有GST的p54重组蛋白;利用亲和层析的方法,富集和纯化带His标签的p54蛋白;以带His标签的p54蛋白为抗原,免疫BALB/C小鼠,制备单克隆抗体;以带有GST标签的p54蛋白为抗原,对制备的单抗进行特异性验证。结果显示:大肠杆菌能高效表达带His标签和GST标签的非洲猪瘟p54蛋白;His标签的p54蛋白免疫BALB/C小鼠后,得到了3株单克隆抗体;Western blot结果表明,3株单克隆抗体能与带有GST标签的p54蛋白相互反应。本研究成功制备了p54的单克隆抗体,为进一步开展非洲猪瘟ELISA、Western blot和细胞免疫荧光检测技术的开发研究奠定了基础。
This study was aimed to obtain the monoclonal antibodies of P54 protein of African swine fever virus(ASFV).Firstly,the outer-membrane segment of p54 gene was amplified.Then the segment was cloned into p ET-30 a plasmid and p GEX 6p-1 plasmid respectively.Plasmids were further transformed into BL21 E.coli,which could express the soluble P54 protein.Using affinity chromatography method,the p54 protein was further purified and used for monoclonal antibodies production.Western blot was used to verify the monoclonal antibodies.As shown in the results,25 kd recombined protein was expressed by E.coli system.Using His specific antibody,Western blot experiment proved that this recombined protein was target p54 protein with his tag.After immunizing the mouse with purified P54 protein,three monoclonal antibodies were obtained.Western blot experiment proved that all three monoclonal antibodies could be reacted with GST-p54 fusion proteins.This study will pave the way for further development of detection methods for ASFV,including ELISA,Western blot and immune-fluorescent assay.
This study was aimed to obtain the monoclonal antibodies of P54 protein of African swine fever virus(ASFV).Firstly,the outer-membrane segment of p54 gene was amplified.Then the segment was cloned into p ET-30 a plasmid and p GEX 6p-1 plasmid respectively.Plasmids were further transformed into BL21 E.coli,which could express the soluble P54 protein.Using affinity chromatography method,the p54 protein was further purified and used for monoclonal antibodies production.Western blot was used to verify the monoclonal antibodies.As shown in the results,25 kd recombined protein was expressed by E.coli system.Using His specific antibody,Western blot experiment proved that this recombined protein was target p54 protein with his tag.After immunizing the mouse with purified P54 protein,three monoclonal antibodies were obtained.Western blot experiment proved that all three monoclonal antibodies could be reacted with GST-p54 fusion proteins.This study will pave the way for further development of detection methods for ASFV,including ELISA,Western blot and immune-fluorescent assay.
引文
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[11]龚振华,王丽萍,臧京帅,等.非洲猪瘟病毒p54蛋白的高效表达及在ELISA中的应用[J].畜牧兽医学报,2013,44(11):1832-1837.
[12]梁瑾,王靖飞.B细胞表位预测方法研究进展[J].Chinese Bulletin of Life Sciences,2009,21(2):320-322.
[2]Dixon L K,Chapman D A G,Netherton C L,et al.African swine fever virus replication and genomics[J].Virus research,2013,173(1):3-14.
[3]Malogolovkin A,Burmakina G,Titov I,et al.Comparative analysis of African swine fever virus genotypes and serogroups[J].Emerging infectious diseases,2015,21(2):312.
[4]Costard S,Mur L,Lubroth J,et al.Epidemiology of African swine fever virus[J].Virus Research,2013,173(1):191-197.
[5]Anderson E C,Hutchings G H,Mukarati N,et al.African swine fever virus infection of the bushpig(Potamochoerusporcus)and its significance in the epidemiology of the disease[J].Veterinary microbiology,1998,62(1):1-15.
[6]Hernáez B,D?az-Gil G,Garc?a-Gallo M,et al.The African swine fever virus dynein-binding protein p54 induces infected cell apoptosis[J].FEBS letters,2004,569(1):224-228.
[7]Rodriguez F,Ley V,Gómez-Puertas P,et al.The structural protein p54 is essential for African swine fever virus viability[J].Virus research,1996,40(2):161-167.
[8]O'Donnell V,Holinka L G,Krug P W,et al.African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL(B119L),when Administered at Low Doses,Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge[J].Journal of Virology,2015,89(16):8556-8566.
[9]Neilan J G,Zsak L,Lu Z,et al.Neutralizing antibodies to African swine fever virus proteins p30,p54,and p72 are not sufficient for antibody-mediated protection[J].Virology,2004,319(2):337-342.
[10]Hou Y,Dong Z,Zhao X,et al.Prokaryotic expression of African swine fever virus P54 gene[J].Chinese Veterinary Science,2009(4):8.
[11]龚振华,王丽萍,臧京帅,等.非洲猪瘟病毒p54蛋白的高效表达及在ELISA中的应用[J].畜牧兽医学报,2013,44(11):1832-1837.
[12]梁瑾,王靖飞.B细胞表位预测方法研究进展[J].Chinese Bulletin of Life Sciences,2009,21(2):320-322.