鲤疱疹病毒Ⅱ型(CyHV-2) ORF25蛋白的原核表达及单克隆抗体的制备
|
摘要
利用PCR方法扩增鲤疱疹病毒Ⅱ型(CyHV-2)ORF25基因的部分基因序列,并将扩增产物连接到原核表达载体pGEX-KG,成功构建了CyHV-2-ORF25-KG重组质粒。IPTG将其诱导后,SDS-PAGE分析表明:目的蛋白得到表达,分子质量大小为42ku,且主要以包涵体形式存在;用纯化的融合蛋白免疫BALB/c小鼠,多次免疫后通过细胞融合,筛选出1株单抗5C6;经间接免疫荧光试验和Western blot试验验证,这株单抗可以识别CyHV-2ORF25蛋白。
The cyprinid herpesvirusⅡ(CyHV-2)ORF25gene mainly encode membrane proteins,which show good antigenicity by antigen eptitope prediction.In this study,ORF25 gene segment was amplified by PCR and cloned into the prokaryotic expression vector pGEX-KG.The CyHV-2-ORF25-KG fusion protein(42ku)was expressed highly under induction of IPTG in the E.coli BL21,and this fusion protein was mainly expressed in the insoluble composition.One monoclonal antibody(McAb 5C6)against ORF25 protein were generated by fusion of mouse myeloma cell line SP2/0and spleen lymphocytes from immunized mice.The McAb 5C6 was further characterized by IFA(indirect immunofluorescent assay)and Western blot assay.This study laid a foundation for the research of the pathogenesis and the detection method of CyHV-2.
The cyprinid herpesvirusⅡ(CyHV-2)ORF25gene mainly encode membrane proteins,which show good antigenicity by antigen eptitope prediction.In this study,ORF25 gene segment was amplified by PCR and cloned into the prokaryotic expression vector pGEX-KG.The CyHV-2-ORF25-KG fusion protein(42ku)was expressed highly under induction of IPTG in the E.coli BL21,and this fusion protein was mainly expressed in the insoluble composition.One monoclonal antibody(McAb 5C6)against ORF25 protein were generated by fusion of mouse myeloma cell line SP2/0and spleen lymphocytes from immunized mice.The McAb 5C6 was further characterized by IFA(indirect immunofluorescent assay)and Western blot assay.This study laid a foundation for the research of the pathogenesis and the detection method of CyHV-2.
引文
[1]ZHANG Q Y,GUI J F.Virus genomes and virus-host interactions in aquaculture animals[J].Sci China Life Sci,2015,58:156-169.
[2]GROFF J M,LAPATRA S E,MUNN R J,et al.A viral epizootic in cultured populations of juvenile goldfish due to a putative herpes virus etiology[J].J Vet Diagn Invest,1998,10(4):375-378.
[3]HANSON L,DISHON A,KOTLER M.Herpesviruses that infect fish[J].Viruses,2011,3(11):2160-2191.
[4]DAVISON A J,EBERLE R,EHLERS B,et al.The order Herpesvirales[J].Arch Virol,2009,154(1):171-177.
[5]JUNG S J,MIYAZAKI T.Herpesviral haematopoietic necrosis of goldfish,Carassius auratus(L.)[J].J Fish Dis,1995,18(3):211-220.
[6]WANG L,HE J,LIANG L G,et al.Mass mortality caused by cyprinid herpesvirus 2(CyHV-Ⅱ)in Prussian carp(Carassius gibelio)in China[J].Fish pathology,2012,32(5):164-173.
[7]李莉娟,罗杨志,刘学芹,等.金鱼鲤疱疹病毒Ⅱ型的分子诊断[J].华中农业大学学报,2013,32(1):92-96.
[8]ANDOR D,MARIA B,GYOERGY C,et al.Introduction of the family Alloherpesviridae:the first molecular detection of herpesviruses of cyprinid fish in Hungary[J].Magyar Allatorvosok Lapja,2011,13(3):174-181.
[9]JEFFREY K R,BATEMAN K,BAYLEY A,et al.Isolation of a cyprinid herpesvirus 2from goldfish,Carassius auratus(L),in the UK[J].J Fish Dis,2007,30(11):649-656.
[10]HEDRICK R P,WALTZEK T B,MCDOWELL T S.Susceptibility of koi carp,common carp,goldfish,and goldfish×common carp hybrids to cyprinid herpesvirus-2and herpesvirus-3[J].J Aquat Anim Health,2006,18(1):26-34.
[11]GOODWIN A E,MERRY G E,SADLER J.Detection of the herpesviral hematopoietic necrosis disease agent(cyprinid herpesvirus 2)in moribund and healthy goldfish:validation of a quantitative PCR diagnostic method[J].Dis Aquat Org,2006,69(2/3):137-143.
[12]田飞焱,何俊强,王璐,等.金鱼疱疹病毒性造血器官坏死病研究进展[J].中国动物检疫,2012,29(4):78-80.
[13]ZHOU Y,JIANG N,MA J,et al.Protective immunity in gibel carp,Carassius gibelio of the truncated proteins of cyprinid herpesvirus 2expressed in Pichia pastoris[J].Fish&shellfish immunology,2015,47(2):1024-1031.
[14]周井祥,李新伟,朱霞,等.锦鲤疱疹病毒CJ株ORF25基因的克隆及生物信息学分析[J].中国兽药杂志,2012,46(3):1-4.
[15]王传武,赵德明.用实验动物制备多克隆抗体-免疫方案的优化[J].实验动物科学与管理,2002,19(4):40-43.
[16]WU B,ZHU B,LUO Y,et al.Generation and characterization of monoclonal antibodies against channel catsh virus[J].Hybridoma,2011,30:555-558.
[17]RUAN X,HUANG S,SHAO L,et al.Monoclonal antibodies against NS4Bprotein of Japanese encephalitis virus[J].Monoclon Antib Immunodiagn Immunother,2013,32(6):382-385.
[18]CHEN Z,SHAO L,YE J,et al.Monoclonal antibodies against NS3and NS5proteins of Japanese encephalitis virus[J].Hybridoma,2012,31:137-141.
[19]孔君,刘箐,韩跃武,等.单克隆抗体制备技术的最新进展及应用前景[J].免疫学杂志,2012,27(2):170-173.
[2]GROFF J M,LAPATRA S E,MUNN R J,et al.A viral epizootic in cultured populations of juvenile goldfish due to a putative herpes virus etiology[J].J Vet Diagn Invest,1998,10(4):375-378.
[3]HANSON L,DISHON A,KOTLER M.Herpesviruses that infect fish[J].Viruses,2011,3(11):2160-2191.
[4]DAVISON A J,EBERLE R,EHLERS B,et al.The order Herpesvirales[J].Arch Virol,2009,154(1):171-177.
[5]JUNG S J,MIYAZAKI T.Herpesviral haematopoietic necrosis of goldfish,Carassius auratus(L.)[J].J Fish Dis,1995,18(3):211-220.
[6]WANG L,HE J,LIANG L G,et al.Mass mortality caused by cyprinid herpesvirus 2(CyHV-Ⅱ)in Prussian carp(Carassius gibelio)in China[J].Fish pathology,2012,32(5):164-173.
[7]李莉娟,罗杨志,刘学芹,等.金鱼鲤疱疹病毒Ⅱ型的分子诊断[J].华中农业大学学报,2013,32(1):92-96.
[8]ANDOR D,MARIA B,GYOERGY C,et al.Introduction of the family Alloherpesviridae:the first molecular detection of herpesviruses of cyprinid fish in Hungary[J].Magyar Allatorvosok Lapja,2011,13(3):174-181.
[9]JEFFREY K R,BATEMAN K,BAYLEY A,et al.Isolation of a cyprinid herpesvirus 2from goldfish,Carassius auratus(L),in the UK[J].J Fish Dis,2007,30(11):649-656.
[10]HEDRICK R P,WALTZEK T B,MCDOWELL T S.Susceptibility of koi carp,common carp,goldfish,and goldfish×common carp hybrids to cyprinid herpesvirus-2and herpesvirus-3[J].J Aquat Anim Health,2006,18(1):26-34.
[11]GOODWIN A E,MERRY G E,SADLER J.Detection of the herpesviral hematopoietic necrosis disease agent(cyprinid herpesvirus 2)in moribund and healthy goldfish:validation of a quantitative PCR diagnostic method[J].Dis Aquat Org,2006,69(2/3):137-143.
[12]田飞焱,何俊强,王璐,等.金鱼疱疹病毒性造血器官坏死病研究进展[J].中国动物检疫,2012,29(4):78-80.
[13]ZHOU Y,JIANG N,MA J,et al.Protective immunity in gibel carp,Carassius gibelio of the truncated proteins of cyprinid herpesvirus 2expressed in Pichia pastoris[J].Fish&shellfish immunology,2015,47(2):1024-1031.
[14]周井祥,李新伟,朱霞,等.锦鲤疱疹病毒CJ株ORF25基因的克隆及生物信息学分析[J].中国兽药杂志,2012,46(3):1-4.
[15]王传武,赵德明.用实验动物制备多克隆抗体-免疫方案的优化[J].实验动物科学与管理,2002,19(4):40-43.
[16]WU B,ZHU B,LUO Y,et al.Generation and characterization of monoclonal antibodies against channel catsh virus[J].Hybridoma,2011,30:555-558.
[17]RUAN X,HUANG S,SHAO L,et al.Monoclonal antibodies against NS4Bprotein of Japanese encephalitis virus[J].Monoclon Antib Immunodiagn Immunother,2013,32(6):382-385.
[18]CHEN Z,SHAO L,YE J,et al.Monoclonal antibodies against NS3and NS5proteins of Japanese encephalitis virus[J].Hybridoma,2012,31:137-141.
[19]孔君,刘箐,韩跃武,等.单克隆抗体制备技术的最新进展及应用前景[J].免疫学杂志,2012,27(2):170-173.