非洲猪瘟病毒主要抗原p54-1的原核表达及多克隆抗体的制备与鉴定
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摘要
为进一步深入研究非洲猪瘟病毒(African swine fever virus,ASFV)p54蛋白的主要抗原表位及抗原性质,本试验根据GenBank中p54基因序列(登录号:NC_001659.2)设计表达区域特异性引物,PCR扩增后连接表达载体pGEX6p-1构建pGEX6p-1-p54-1原核表达质粒;将该质粒转化大肠杆菌BL21感受态细胞,经IPTG诱导,SDS-PAGE鉴定融合蛋白的表达,切除GST标签后采用阴离子柱纯化目的蛋白并鉴定;将纯化蛋白与佐剂混合乳化后作为免疫原,免疫小鼠制备p54-1蛋白多克隆抗体;采用ELISA和Western blotting检测抗体的效价和反应特性。结果显示,重组p54-1融合蛋白以可溶形式表达,切除标签后的纯化蛋白能够与ASFV阳性血清发生反应,而与PRRSV和PCV3不发生反应。利用该蛋白免疫获得的多克隆抗体经ELISA检测其抗体效价为1∶128 000。Western blotting结果显示,制备的多克隆抗体能够识别ASFV p54蛋白。表明本研究成功获得了较高纯度的p54-1蛋白,制备的p54-1多克隆抗体具有较高反应性和特异性,为后续非洲猪瘟双抗夹心ELISA检测方法的建立提供依据。
In order to further study the major epitopes and antigen properties of African swine fever virus(ASFV)p54-1 protein,specific primers were designed according to p54 gene sequence retrieved from GenBank(accession No.:NC_001659.2),the target sequence of encoding p54-1 protein was amplified by PCR.Then it was ligated into pGEX6 p-1 vector and constructed prokaryotic expression plasmid(pGEX6 p-1-p54-1).The plasmid was transfected into E.coli BL21,and the expression of recombinant protein was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The fusion protein cleaved the GST-tag using thrombin,and then purified by anion exchange column.The protein was identified by ELISA and emulsify with adju-vant,the prepared immunogen was inoculated into mouse to prepare of p54-1 protein specific polyclonal antibody.The immune-activity and titers of the prepared polyclonal antibody were determined by ELISA and Western blotting.The results showed that the expressed recombinant protein p54-1 existed in a soluble form.The p54-1 protein cleaved GST-tag could react with the positive serum of ASFV,but no with negative serum,PRRSV and PCV3.The prepared polyclonal antibody titer was 1∶128 000.Western blotting result demonstrated that the prepared polyclonal antibody could recognize the p54 protein.In conclusion,the high purified expressed protein of ASFV p54-1 had been successfully prepared and p54-1 specific polyclonal antibody showed wonderful immunocompetence and specificity,providing foundation for the development of sandwich ELISA detection method of ASF.
In order to further study the major epitopes and antigen properties of African swine fever virus(ASFV)p54-1 protein,specific primers were designed according to p54 gene sequence retrieved from GenBank(accession No.:NC_001659.2),the target sequence of encoding p54-1 protein was amplified by PCR.Then it was ligated into pGEX6 p-1 vector and constructed prokaryotic expression plasmid(pGEX6 p-1-p54-1).The plasmid was transfected into E.coli BL21,and the expression of recombinant protein was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The fusion protein cleaved the GST-tag using thrombin,and then purified by anion exchange column.The protein was identified by ELISA and emulsify with adju-vant,the prepared immunogen was inoculated into mouse to prepare of p54-1 protein specific polyclonal antibody.The immune-activity and titers of the prepared polyclonal antibody were determined by ELISA and Western blotting.The results showed that the expressed recombinant protein p54-1 existed in a soluble form.The p54-1 protein cleaved GST-tag could react with the positive serum of ASFV,but no with negative serum,PRRSV and PCV3.The prepared polyclonal antibody titer was 1∶128 000.Western blotting result demonstrated that the prepared polyclonal antibody could recognize the p54 protein.In conclusion,the high purified expressed protein of ASFV p54-1 had been successfully prepared and p54-1 specific polyclonal antibody showed wonderful immunocompetence and specificity,providing foundation for the development of sandwich ELISA detection method of ASF.
引文
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[18]梁云浩,曹琛福,陶虹,等.非洲猪瘟病毒p54蛋白的真核表达及间接ELISA检测方法的建立[J].中国兽医科学,2014,44(4):373-378.LIANG Y H,CAO C F,TAO H,et al.Eukaryotic expression of African swine fever virus p54protein and development of an indirect ELISA for detection of antibody against ASFV[J].Chinese Veterinary Science,2014,44(4):373-378.(in Chinese)
[19]曹琛福,梁云浩,陶虹,等.非洲猪瘟病毒p54基因的原核表达及其抗体的间接ELISA检测方法的建立[J].动物医学进展,2014,35(2):6-10.CAO C F,LIANG Y H,TAO H,et al.Prokaryotic expression of p54gene of African swine fever virus and establishment of indirect ELISA for detection of its antibody against p54[J].Progress in Veterinary Medicine,2014,35(2):6-10.(in Chinese)
[20]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.GONG Z H,WANG L P,ZANG J S,et al.Studies on the efficient expression of African swine fever virus p54protein and its antigenic analysis in ELISA[J].Acta Veterinaria et Zootechnica Sinica,2013,44(11):1832-1837.(in Chinese)
[21] TAKAMATSU H H,DENYER M S,LACASTA A,et al.Cellular immunity in ASFV response[J].Virus Research,2013,173(1):110-121.
[22]曹琛福,梁云浩,花群俊,等.非洲猪瘟病毒p54蛋白抗原表位预测及鉴定[J].中国预防兽医学报,2014,36(11):848-851.CAO C F,LIANG Y H,HUA Q J,et al.B cell epitope prediction and identification on the p54of African swine fever virus[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(11):848-851.(in Chinese)
[23]曹琛福,梁云浩,吕建强,等.非洲猪瘟病毒p54蛋白特定抗原表位单克隆抗体的制备[J].中国兽医科学,2015,45(1):75-79.CAO C F,LIANG Y H,LV J Q,et al.Preparation of monoclonal antibody against specific antigenic epitope of the p54protein of African swine fever virus[J].Chinese Veterinary Science,2015,45(1):75-79.(in Chinese)
[24]刘祥,陈春琳,王杨科,等.小鼠骨桥蛋白的原核表达及多克隆抗体的制备与鉴定[J].中国畜牧兽医,2015,42(5):1069-1075.LIU X,CHEN C L,WANG Y K,et al.Prokaryotic expression,polyclonal antibody preparation and identification of mouse osteopontin protein[J].China Animal Husbandry&Veterinary Medicine,2015,42(5):1069-1075.(in Chinese)
[2] RONISH B,HAKHVERDYAN M,STHL K,et al.Design and verification of a highly reliable linear-after-the-exponential PCR(LATE-PCR)assay for the detection of African swine fever virus[J].Journal of Virological Methods,2011,172(1-2):8-15.
[3]戈胜强,吴晓东,张志诚,等.非洲猪瘟疫苗研究进展[J].畜牧兽医学报,2016,47(1):10-15.GE S Q,WU X D,ZHANG Z C,et al.Progress in development of African swine fever vaccine[J].Acta Veterinaria et Zootechnica Sinica,2016,47(1):10-15.(in Chinese)
[4] REVILLA Y,PREZ-NU'EZ D,RICHT J A.African swine fever virus biology and vaccine approaches[J].Advances in Virus Research,2018,100:41-74.
[5] 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.
[6] MALOGOLOVKIN A,BURMAKINA G,TITOV I,et al.Comparative analysis of African swine fever virus genotypes and serogroups[J].Emerging Infectious Disease,2015,21(2):312-315.
[7]张鑫宇,左伟勇,朱善元,等.非洲猪瘟病毒p54抗体胶体金试纸检测方法的建立[J].中国预防兽医学报,2014,36(4):281-285.ZHANG X Y,ZUO W Y,ZHU S Y,et al.Establishment of colloidal gold strip for detecting antibody against African swine fever virus[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(4):281-285.(in Chinese)
[8]冯春燕,宋晓晖,仇松寅,等.非洲猪瘟病毒p54蛋白的表达及单抗制备[J].中国动物检疫,2016,33(5):80-84.FENG C Y,SONG X H,QIU S Y,et al.Expression and purification of p54protein of Africa swine fever disease and production of its specific monoclonal antibodies[J].China Animal Health Inspection,2016,33(5):80-84.(in Chinese)
[9] RODRIGUEZ J M,GARCIA E.African swine fever virus structural protein p54 is essential for the recruitment of envelope precursors to assembly sites[J].Journal of Virology,2004,78(8):4299-4313.
[10]许兰娇,洪智敏,司微,等.鸡β-防御素-9的融合表达及其多克隆抗体的制备与鉴定[J].动物营养学报,2013,25(3):603-608.XU L J,HONG Z M,SI W,et al.Fusion expression of chickenβ-defensin-9and preparation,characterization of its polyclonal antibody[J].Acta Zoonutrimenta Sinica,2013,25(3):603-608.(in Chinese)
[11]邓俊花,林祥梅,吴绍强.非洲猪瘟研究新进展[J].中国动物检疫,2017,34(8):66-71.DENG J H,LIN X M,WU S Q.Research progress of African swine fever[J].China Animal Health Inspection,2017,34(8):66-71.(in Chinese)
[12] GALLARDO M C,REOYO A T,FERNNDEZPINERO J,et al.African swine fever:A global view of the current challenge[J].Porcine Health Management,2015,1(1):21.
[13] GIMNEZ-LIROLA L G,MUR L,RIVERA B,et al.Detection of African swine fever virus antibodies in oral fluid specimens using a recombinant protein 30(p30)dual matrix indirect ELISA[J].PLoS One,2016,11(9):e0161230.
[14] HEIMERMAN M E,MURGIA M V,WU P,et al.Linear epitopes in African swine fever virus p72recognized by monoclonal antibodies prepared againstbaculovirus-expressed antigen[J].Journal of Veterinary Diagnostic Investigation.2018,30(3):406-412.
[15] GALLARDO C,SOLER A,NIETO R,et al.Comparative evaluation of novel African swine fever virus(ASF)antibody detection techniques derived from specific ASF viral genotypes with the OIE internationally prescribed serological tests[J].Veterinary Microbiology,2013,162(1):32-43.
[16] GALLARDO C,REIS A L,KALEMAZIKUSOKA G,et al.Recombinant antigen targets forserodiagnosis of African swine fever[J].Clinical and Vaccine Immunology,2009,16(7):1012-1020.
[17] PREZ-FILGUEIRA D M,GONZALEZ-CAMACHO F,GALLARDO C,et al.Optimization and validation of recombinant serological tests for African swine fever diagnosis based on detection of the p30protein produced inTrichoplusiani larvae[J].Journal of Clinical Microbiology,2006,44(9):3114-3121.
[18]梁云浩,曹琛福,陶虹,等.非洲猪瘟病毒p54蛋白的真核表达及间接ELISA检测方法的建立[J].中国兽医科学,2014,44(4):373-378.LIANG Y H,CAO C F,TAO H,et al.Eukaryotic expression of African swine fever virus p54protein and development of an indirect ELISA for detection of antibody against ASFV[J].Chinese Veterinary Science,2014,44(4):373-378.(in Chinese)
[19]曹琛福,梁云浩,陶虹,等.非洲猪瘟病毒p54基因的原核表达及其抗体的间接ELISA检测方法的建立[J].动物医学进展,2014,35(2):6-10.CAO C F,LIANG Y H,TAO H,et al.Prokaryotic expression of p54gene of African swine fever virus and establishment of indirect ELISA for detection of its antibody against p54[J].Progress in Veterinary Medicine,2014,35(2):6-10.(in Chinese)
[20]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.GONG Z H,WANG L P,ZANG J S,et al.Studies on the efficient expression of African swine fever virus p54protein and its antigenic analysis in ELISA[J].Acta Veterinaria et Zootechnica Sinica,2013,44(11):1832-1837.(in Chinese)
[21] TAKAMATSU H H,DENYER M S,LACASTA A,et al.Cellular immunity in ASFV response[J].Virus Research,2013,173(1):110-121.
[22]曹琛福,梁云浩,花群俊,等.非洲猪瘟病毒p54蛋白抗原表位预测及鉴定[J].中国预防兽医学报,2014,36(11):848-851.CAO C F,LIANG Y H,HUA Q J,et al.B cell epitope prediction and identification on the p54of African swine fever virus[J].Chinese Journal of Preventive Veterinary Medicine,2014,36(11):848-851.(in Chinese)
[23]曹琛福,梁云浩,吕建强,等.非洲猪瘟病毒p54蛋白特定抗原表位单克隆抗体的制备[J].中国兽医科学,2015,45(1):75-79.CAO C F,LIANG Y H,LV J Q,et al.Preparation of monoclonal antibody against specific antigenic epitope of the p54protein of African swine fever virus[J].Chinese Veterinary Science,2015,45(1):75-79.(in Chinese)
[24]刘祥,陈春琳,王杨科,等.小鼠骨桥蛋白的原核表达及多克隆抗体的制备与鉴定[J].中国畜牧兽医,2015,42(5):1069-1075.LIU X,CHEN C L,WANG Y K,et al.Prokaryotic expression,polyclonal antibody preparation and identification of mouse osteopontin protein[J].China Animal Husbandry&Veterinary Medicine,2015,42(5):1069-1075.(in Chinese)