PPRV武威分离株N基因的原核表达及免疫原性分析
|
摘要
小反刍兽疫(Peste des petits ruminants, PPR)是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性传染病,严重危害世界养羊业的健康发展。2013年以来,国内多地出现小反刍兽疫疫情,给我国养羊业造成了重大经济损失。本研究根据小反刍兽疫病毒Nigeria 75/1株N基因核苷酸序列(GenBank No. HQ197753)设计特异性引物,扩增获得PPRV武威分离株N基因全序列(GenBank No. KY429031),在序列分析的基础上,构建该基因的原核表达载体pET-PPRV-N,并将其转化大肠杆菌(Escherichia coli) Rosetta (DE3)后诱导表达。纯化表达产物,免疫Balb/C小鼠(Mus musculus)制备多克隆抗体,进而应用ELISA、Western blot和免疫荧光检测(immunological fluorescence assay, IFA)对PPRV N蛋白的免疫原性进行分析。结果表明,PPRV武威分离株属于Ⅳ系,且其N基因全序列与国内24株PPRV分离株及2016蒙古株的同源性高达99.0%;重组蛋白His-PPRV-N在大肠杆菌Rosetta (DE3)中成功获得表达,其相对分子质量约为57.8 kD,且主要以可溶性形式存在;ELISA、Western blot及IFA结果显示,重组蛋白小鼠多抗ELISA效价可达1∶64 000,可与PPRV疫苗株或武威分离株的N蛋白特异性结合,且制备的多抗显示出对PPRV明显的中和作用,表明了重组蛋白良好的免疫原性。本研究结果为PPRV检测方法的建立和N蛋白生物学功能的进一步研究提供了基础资料。
Peste des petits ruminants(PPR) is an acute infectious disease caused by Peste des petits ruminants virus(PPRV), which seriously endangers the healthy development of the world's sheep industry. Since 2013,PPR has been epidemic in many places in China, and the sheep industry suffered huge losses. In this study, the specific primers were designed according to the complete sequence of N gene of PPRV Nigeria 75/1 strain(GenBank No. HQ197753) in GenBank, and the full-length sequence of N gene of PPRV Wuwei strain(GenBank No. KY429031) was amplified. Based on sequencing and sequence analysis, the prokaryotic expression vector pET-PPRV-N was constructed and then was transformed into Escherichia coli Rosetta(DE3).Subsequently, the recombinant protein His-PPRV-N was purified and the Balb/C mice(Mus musculus) were immunized. Then the anti-serum against N protein of PPRV was prepared. Finally, the immunogenicity of recombinant protein was analysed by ELISA, Western blot and IFA(immunological fluorescence assay). The results showed that the N gene of PPRV Wuwei strain shared 99.0% homology with 24 PPRV strains isolated from China as well as Mongolia strain 2016. The phylogenetic tree analysis identified that the PPRV Wuwei strain belonged to lineage Ⅳ. The result of SDS-PAGE showed that the recombinant protein His-PPRV-N was successfully expressed in E. coli Rosetta(DE3) with the form of soluble protein, and the relative molecular weight was approximately 57.8 kD. The results of ELISA,Western blot and IFA indicated that the His-PPRVN possessed excellent immunogenicity. The results provide basic information for the establishment of the detection method of PPRV and for further study on biological functions of PPRV N protein.
Peste des petits ruminants(PPR) is an acute infectious disease caused by Peste des petits ruminants virus(PPRV), which seriously endangers the healthy development of the world's sheep industry. Since 2013,PPR has been epidemic in many places in China, and the sheep industry suffered huge losses. In this study, the specific primers were designed according to the complete sequence of N gene of PPRV Nigeria 75/1 strain(GenBank No. HQ197753) in GenBank, and the full-length sequence of N gene of PPRV Wuwei strain(GenBank No. KY429031) was amplified. Based on sequencing and sequence analysis, the prokaryotic expression vector pET-PPRV-N was constructed and then was transformed into Escherichia coli Rosetta(DE3).Subsequently, the recombinant protein His-PPRV-N was purified and the Balb/C mice(Mus musculus) were immunized. Then the anti-serum against N protein of PPRV was prepared. Finally, the immunogenicity of recombinant protein was analysed by ELISA, Western blot and IFA(immunological fluorescence assay). The results showed that the N gene of PPRV Wuwei strain shared 99.0% homology with 24 PPRV strains isolated from China as well as Mongolia strain 2016. The phylogenetic tree analysis identified that the PPRV Wuwei strain belonged to lineage Ⅳ. The result of SDS-PAGE showed that the recombinant protein His-PPRV-N was successfully expressed in E. coli Rosetta(DE3) with the form of soluble protein, and the relative molecular weight was approximately 57.8 kD. The results of ELISA,Western blot and IFA indicated that the His-PPRVN possessed excellent immunogenicity. The results provide basic information for the establishment of the detection method of PPRV and for further study on biological functions of PPRV N protein.
引文
冯向辉,陈三民,孔汉金,等.2017.小反刍兽疫病毒重组N蛋白抗原制备及间接ELISA检测方法的建立[J].畜牧与兽医,49(12):75-79.(Feng X H,Cheng S M,Kong HJ,et al.2017.Preparation of recombinant N protein antigen of Peste des petits ruminant virus and establishment of indirect ELISA method[J].Animal Husbandry and Veterinary Medicine,49(12):75-79.)
龙云凤,周晓黎,杨俊兴,等.2012.小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用[J].畜牧兽医学报,43(4):588-595.(Long Y F,Zhou X L,Yang J X,et al.2012.Preparation and applicationof monoclonal antibodies to nucleocapsid proteins of PPRV[J].Chinese Journal of Animal and Veterinary Sciences,43(4):588-595.)
庞文静,付明哲,张琪,等.2017.基于N蛋白的PPRV间接ELISA检测方法的建立及应用[J].西北农林科技大学学报:自然科学版,45(11):1-8.(Pang W J,Fu M Z,Zhang Q,et al.2017.Establishment and application of indirect ELISA detection method based on N protein of PPRV[J].Journal of Northwest A&F University:Natural Science Edition,45(11):1-8.)
闫飞虎,王化磊,邱泊宁,等.2016.小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备[J].中国生物制品学杂志,29(6):576-581.(Yan F H,Wang H L,Qiu B N,et al.2016.Prokaryotic expression of H protein of Peste des petits ruminants virus and preparation of polyclonal antibody[J].Chinese Journal of Biologicals,29(6):576-581.)
向蓉,黄勉,向华,等.2017.小反刍兽疫流行状态及检测方法研究进展[J].中国动物检疫,34(10):46-49.(Xiang R,Huang M,Xiang H,et al.2017.Epidemic status of PPRV and research progress in detection methods[J].China Animal Health Inspection,34(10):46-49.)
吴锦艳,尚佑军,田宏,等.2016.2007-2014年国内外小反刍兽疫流行现状及分析[J].中国兽医学报,36(4):687-693.(Wu J Y,Shang Y J,Tian H,et al.2016.The current situation and analysis of PPRV in the world from2007 to 2014[J].Chinese Journal of Veterinary Science,36(4):687-693.)
王净,王鹏,王长顺.2017.小反刍兽疫病毒N基因部分序列分子演化规律[J].中国兽医学报,37(12):2304-2309.(Wang J,Wang P,Wang C S,et al.2017.Molecular evolution of the partial sequence of the N gene of PPRV[J].Chinese Journal of Veterinary Science,37(12):2304-2309.)
张喜悦,王志亮,刘春菊,等.2008.小反刍兽疫病毒(PPRV)N蛋白的表达与ELISA检测方法的建立[J].中国兽医学报,28(2):146-148.(Zhang X Y,Wang Z L,Liu C J,et al.2008.Expression of N protein of PPRV and establishment of indirect ELISA method[J].Chinese Journal of Veterinary Science,28(2):146-148.)
Banyard A C,Parida S,Batten C,et al.2010.Global distribution of Peste des petits ruminants virus and prospects for improved diagnosis and control[J].Journal of General Virology,91(Pt 12):2885-2897.
Diallo A.1990.Morbillivirus group:Genome organisation and proteins[J].Veterinary Microbiology,231(1-4):155.
Dechamma H J,Dighe V,Kumar C A,et al.2006.Identification of T-helper and linear B epitope in the hypervariable region of nucleocapsid protein of PPRV and its use in the development of specific antibodies to detect viral antigen[J].Veterinary Microbiology,118(4):201-211.
Grodzicker T,Lewis J B,Anderson C W.1990.Peste des petits ruminants[J].Revue Scientifique Et Technique,9(4):935.
Kwiatek O,Minet C,Grillet C,et al.2007.Peste des petits ruminants(PPR)outbreak in Tajikistan[J].Journal of Comparative Pathology,136(2):111-119.
Minet C,Yami M,Egzabhier B,et al.2009.Sequence analysis of the large(L)polymerase gene and trailer of the Peste des petits ruminants virus vaccine strain Nigeria 75/1:Expression and use of the L protein in reverse genetics[J].Virus Research,145(1):9-17.
Shaila M S,Shamaki D,Forsyth M A,et al.1996.Geographic distribution and epidemiology of Peste des petits rumi?nants virus[J].Virus Research,43(2):149-53.
Wu X,Li L,Li J,et al.2016.Peste des Petits Ruminants Virus?es re-emerging in China,2013-2014.[J].Transboundary&Emerging Diseases,63(5):e441-e446.
Wang J,Miao W,Wang S,et al.2015.Peste des petits rumi?nants virus in Heilongjiang province,China,2014[J].Emerging Infectious Diseases,21(4):677-680.
Zahur A B,Ullah A,Hussain M,et al.2009.Sero-epidemiology of Peste des petits ruminants(PPR)in Pakistan[J].Preventive Veterinary Medicine,29(4):87-92.
Zhou X Y,Wang Y,Zhu J,et al.2018.First report of Peste des petits ruminants virus lineageⅡin hydropotesinermis,China[J].Transboundary&Emerging Diseases,65(3):205-209.
龙云凤,周晓黎,杨俊兴,等.2012.小反刍兽疫病毒核蛋白单克隆抗体的制备与初步应用[J].畜牧兽医学报,43(4):588-595.(Long Y F,Zhou X L,Yang J X,et al.2012.Preparation and applicationof monoclonal antibodies to nucleocapsid proteins of PPRV[J].Chinese Journal of Animal and Veterinary Sciences,43(4):588-595.)
庞文静,付明哲,张琪,等.2017.基于N蛋白的PPRV间接ELISA检测方法的建立及应用[J].西北农林科技大学学报:自然科学版,45(11):1-8.(Pang W J,Fu M Z,Zhang Q,et al.2017.Establishment and application of indirect ELISA detection method based on N protein of PPRV[J].Journal of Northwest A&F University:Natural Science Edition,45(11):1-8.)
闫飞虎,王化磊,邱泊宁,等.2016.小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备[J].中国生物制品学杂志,29(6):576-581.(Yan F H,Wang H L,Qiu B N,et al.2016.Prokaryotic expression of H protein of Peste des petits ruminants virus and preparation of polyclonal antibody[J].Chinese Journal of Biologicals,29(6):576-581.)
向蓉,黄勉,向华,等.2017.小反刍兽疫流行状态及检测方法研究进展[J].中国动物检疫,34(10):46-49.(Xiang R,Huang M,Xiang H,et al.2017.Epidemic status of PPRV and research progress in detection methods[J].China Animal Health Inspection,34(10):46-49.)
吴锦艳,尚佑军,田宏,等.2016.2007-2014年国内外小反刍兽疫流行现状及分析[J].中国兽医学报,36(4):687-693.(Wu J Y,Shang Y J,Tian H,et al.2016.The current situation and analysis of PPRV in the world from2007 to 2014[J].Chinese Journal of Veterinary Science,36(4):687-693.)
王净,王鹏,王长顺.2017.小反刍兽疫病毒N基因部分序列分子演化规律[J].中国兽医学报,37(12):2304-2309.(Wang J,Wang P,Wang C S,et al.2017.Molecular evolution of the partial sequence of the N gene of PPRV[J].Chinese Journal of Veterinary Science,37(12):2304-2309.)
张喜悦,王志亮,刘春菊,等.2008.小反刍兽疫病毒(PPRV)N蛋白的表达与ELISA检测方法的建立[J].中国兽医学报,28(2):146-148.(Zhang X Y,Wang Z L,Liu C J,et al.2008.Expression of N protein of PPRV and establishment of indirect ELISA method[J].Chinese Journal of Veterinary Science,28(2):146-148.)
Banyard A C,Parida S,Batten C,et al.2010.Global distribution of Peste des petits ruminants virus and prospects for improved diagnosis and control[J].Journal of General Virology,91(Pt 12):2885-2897.
Diallo A.1990.Morbillivirus group:Genome organisation and proteins[J].Veterinary Microbiology,231(1-4):155.
Dechamma H J,Dighe V,Kumar C A,et al.2006.Identification of T-helper and linear B epitope in the hypervariable region of nucleocapsid protein of PPRV and its use in the development of specific antibodies to detect viral antigen[J].Veterinary Microbiology,118(4):201-211.
Grodzicker T,Lewis J B,Anderson C W.1990.Peste des petits ruminants[J].Revue Scientifique Et Technique,9(4):935.
Kwiatek O,Minet C,Grillet C,et al.2007.Peste des petits ruminants(PPR)outbreak in Tajikistan[J].Journal of Comparative Pathology,136(2):111-119.
Minet C,Yami M,Egzabhier B,et al.2009.Sequence analysis of the large(L)polymerase gene and trailer of the Peste des petits ruminants virus vaccine strain Nigeria 75/1:Expression and use of the L protein in reverse genetics[J].Virus Research,145(1):9-17.
Shaila M S,Shamaki D,Forsyth M A,et al.1996.Geographic distribution and epidemiology of Peste des petits rumi?nants virus[J].Virus Research,43(2):149-53.
Wu X,Li L,Li J,et al.2016.Peste des Petits Ruminants Virus?es re-emerging in China,2013-2014.[J].Transboundary&Emerging Diseases,63(5):e441-e446.
Wang J,Miao W,Wang S,et al.2015.Peste des petits rumi?nants virus in Heilongjiang province,China,2014[J].Emerging Infectious Diseases,21(4):677-680.
Zahur A B,Ullah A,Hussain M,et al.2009.Sero-epidemiology of Peste des petits ruminants(PPR)in Pakistan[J].Preventive Veterinary Medicine,29(4):87-92.
Zhou X Y,Wang Y,Zhu J,et al.2018.First report of Peste des petits ruminants virus lineageⅡin hydropotesinermis,China[J].Transboundary&Emerging Diseases,65(3):205-209.