非洲猪瘟病毒沈阳分离株p30基因的克隆及其亲水区的原核表达
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摘要
2018年中国首次发生了非洲猪瘟疫情。本研究从来源于沈阳地区的1个非洲猪瘟病毒(African swine fever vi?rus, ASFV)分离株中克隆了p30基因(GenBank No. MK482370)。序列分析发现,该基因与安徽分离株相同(GenBank No.MK128995),而与已报道的沈阳分离株SY-18株(GenBank No. MH766894)不同,新克隆的p30基因全长585 bp,编码194个氨基酸,而SY-18株p30基因全长606 bp,编码201个氨基酸。该结果提示,我国的ASFV存在基因变异或者传播来源复杂,流行病学分析应关注该基因,不同毒株的致病力有待深入研究。本研究利用pGEX-6p-1表达系统成功表达了p30基因的亲水区;通过Ni-NTA-琼脂糖亲和层析技术对重组蛋白进行纯化,获得了纯度较高的重组蛋白,为ASFV的血清学监测提供了抗原。
African swine fever outbroke in China in 2018. The p30 gene(GenBank No. MK482370) was cloned from an African Swine Fever Virus(ASFV) strain isolated in Shenyang. It has 100% nucleotide identity with Anhui isolated strain(GenBank No. MK128995). However, there is some difference with reported SY-18(GenBank No.MH766894). It has 585 base pairs(bp) coding 194 amino acids(aa), while the p30 gene of SY-18 has 606 bp coding201 aa. This observation suggested that variation occurred in ASFV of China, whether the virulence varies remains to be further studied. Plasmid pGEX-6 p-1 were used in this study to express the hydrophilic area of p30 gene, and the recombinant proteins were purified by Ni-NTA-agarose affinity chromatography. This study provided antigen for ASFV serologic surveillance.
African swine fever outbroke in China in 2018. The p30 gene(GenBank No. MK482370) was cloned from an African Swine Fever Virus(ASFV) strain isolated in Shenyang. It has 100% nucleotide identity with Anhui isolated strain(GenBank No. MK128995). However, there is some difference with reported SY-18(GenBank No.MH766894). It has 585 base pairs(bp) coding 194 amino acids(aa), while the p30 gene of SY-18 has 606 bp coding201 aa. This observation suggested that variation occurred in ASFV of China, whether the virulence varies remains to be further studied. Plasmid pGEX-6 p-1 were used in this study to express the hydrophilic area of p30 gene, and the recombinant proteins were purified by Ni-NTA-agarose affinity chromatography. This study provided antigen for ASFV serologic surveillance.
引文
[1]王志亮.非洲猪瘟研究进展[J].中国动物检疫,2011,28(6):70-74.
[2]王志刚,李振,齐晓亮.掀开非洲猪瘟的“面纱”[J].兽医导刊,2018(17):17-19.
[3]王华,王君玮,徐天刚,等.非洲猪瘟流行病学和诊断方法的研究进展[J].中国兽医科学,2008,38(6):544-548.
[4]Dela Torre A,Bosch J,Iglesias I,et al.Assessing the risk of African swine fever introduction into the European Union by wild boar[J].Transboundary and emerging diseases,2015,62(3):272-279.
[5]Montgomery R E.On a form of swine fever occurring in British East Africa(Kenya Colony)[J].Journal of comparative pathology and therapeutics,1921,34:159-191.
[6]殷震,刘景华.动物病毒学[M].北京:科学出版社,1997:1197-1205.
[7]洪健,周雪平.ICTV第八次报告的最新病毒分类系统[J].中国病毒学,2006,21(1):84-96.
[8]侯艳梅,董志珍,赵祥平,等.非洲猪瘟病毒P54基因原核表达载体的构建与表达[J].中国兽医科学,2009,39(4):316-320.
[9]Yanez R J,Rodr??guez J M,Nogal M L,et al.Analysis of the complete nucleotide sequence of African swine fever virus[J].Virology,1995,208(1):249-278.
[10]Chapman D A G,Tcherepanov V,Upton C,et al.Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates[J].Journal of General Virology,2008,89(2):397-408.
[11]Portugal R,Coelho J,H?per D,et al.Related strains of African swine fever virus with different virulence:genome comparison and analysis[J].Journal of General Virology,2015,96(2):408-419.
[12]Galindo I,Alonso C.African swine fever virus:a review[J].Viruses,2017,9(5):103.
[13]Sánchez E G,Pérez-Nú?ez D,Revilla Y.Mechanisms of Entry and Endosomal Pathway of African Swine Fever Virus[J].Vaccines,2017,5(4):42.
[14]欧云文,阎传忠,张杰,等.非洲猪瘟病毒的分子病原学及致病机理研究进展[J].中国畜牧兽医,2017,44(7):2139-2146.
[15]Hernaez B,Escribano J M,Alonso C.African swine fever virus protein p30 interaction with heterogeneous nuclear ribonucleoprotein K(hnRNP-K)during infection[J].FEBS letters,2008,582(23-24):3275-3280.
[16]Afonso C L,Alcaraz C,Brun A,et al.Characterization of p30,a highly antigenic membrane and secreted protein of African swine fever virus[J].Virology,1992,189(1):368-373.
[17]Oviedo J M,Rodriguez F,Gomez-Puertas P,et al.High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents[J].Journal of virological methods,1997,64(1):27-36.
[18]包雨鑫,丁军莉,郑海英,等.非洲猪瘟研究进展[J].黑龙江畜牧兽医,2018(24):78-80.
[19]Arias M,Dela Torre A,Dixon L,et al.Approaches and perspectives for development of African swine fever virus vaccines[J].Vaccines,2017,5(4):35.
[20]Cubillos C,Gómez-Sebastian S,Moreno N,et al.African swine fever virus serodiagnosis:a general review with a focus on the analyses of African serum samples[J].Virus research,2013,173(1):159-167.
[21]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.
[22]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.
[23]Tabares E,Fernandez M,Salvador-Temprano E,et al.A reliable enzyme linked immunosorbent assay for African swine fever using the major structural protein as antigenic reagent[J].Archives of virology,1981,70(3):297-300.
[24]Gallardo C,Blanco E,Rodríguez J M,et al.Antigenic properties and diagnostic potential of African swine fever virus protein pp62expressed in insect cells[J].Journal of clinical microbiology,2006,44(3):950-956.
[25]Tulman E R,Rock D L.Novel virulence and host range genes of African swine fever virus[J].Current opinion in microbiology,2001,4(4):456-461.
[26]Perez-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 p30 protein produced in Trichoplusia ni larvae[J].Journal of Clinical Microbiology,2006,44(9):3114-3121.
[27]储德文.非洲猪瘟病毒p30重组蛋白的表达及单克隆抗体制备[D].扬州:扬州大学,2013.
[2]王志刚,李振,齐晓亮.掀开非洲猪瘟的“面纱”[J].兽医导刊,2018(17):17-19.
[3]王华,王君玮,徐天刚,等.非洲猪瘟流行病学和诊断方法的研究进展[J].中国兽医科学,2008,38(6):544-548.
[4]Dela Torre A,Bosch J,Iglesias I,et al.Assessing the risk of African swine fever introduction into the European Union by wild boar[J].Transboundary and emerging diseases,2015,62(3):272-279.
[5]Montgomery R E.On a form of swine fever occurring in British East Africa(Kenya Colony)[J].Journal of comparative pathology and therapeutics,1921,34:159-191.
[6]殷震,刘景华.动物病毒学[M].北京:科学出版社,1997:1197-1205.
[7]洪健,周雪平.ICTV第八次报告的最新病毒分类系统[J].中国病毒学,2006,21(1):84-96.
[8]侯艳梅,董志珍,赵祥平,等.非洲猪瘟病毒P54基因原核表达载体的构建与表达[J].中国兽医科学,2009,39(4):316-320.
[9]Yanez R J,Rodr??guez J M,Nogal M L,et al.Analysis of the complete nucleotide sequence of African swine fever virus[J].Virology,1995,208(1):249-278.
[10]Chapman D A G,Tcherepanov V,Upton C,et al.Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates[J].Journal of General Virology,2008,89(2):397-408.
[11]Portugal R,Coelho J,H?per D,et al.Related strains of African swine fever virus with different virulence:genome comparison and analysis[J].Journal of General Virology,2015,96(2):408-419.
[12]Galindo I,Alonso C.African swine fever virus:a review[J].Viruses,2017,9(5):103.
[13]Sánchez E G,Pérez-Nú?ez D,Revilla Y.Mechanisms of Entry and Endosomal Pathway of African Swine Fever Virus[J].Vaccines,2017,5(4):42.
[14]欧云文,阎传忠,张杰,等.非洲猪瘟病毒的分子病原学及致病机理研究进展[J].中国畜牧兽医,2017,44(7):2139-2146.
[15]Hernaez B,Escribano J M,Alonso C.African swine fever virus protein p30 interaction with heterogeneous nuclear ribonucleoprotein K(hnRNP-K)during infection[J].FEBS letters,2008,582(23-24):3275-3280.
[16]Afonso C L,Alcaraz C,Brun A,et al.Characterization of p30,a highly antigenic membrane and secreted protein of African swine fever virus[J].Virology,1992,189(1):368-373.
[17]Oviedo J M,Rodriguez F,Gomez-Puertas P,et al.High level expression of the major antigenic African swine fever virus proteins p54 and p30 in baculovirus and their potential use as diagnostic reagents[J].Journal of virological methods,1997,64(1):27-36.
[18]包雨鑫,丁军莉,郑海英,等.非洲猪瘟研究进展[J].黑龙江畜牧兽医,2018(24):78-80.
[19]Arias M,Dela Torre A,Dixon L,et al.Approaches and perspectives for development of African swine fever virus vaccines[J].Vaccines,2017,5(4):35.
[20]Cubillos C,Gómez-Sebastian S,Moreno N,et al.African swine fever virus serodiagnosis:a general review with a focus on the analyses of African serum samples[J].Virus research,2013,173(1):159-167.
[21]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.
[22]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.
[23]Tabares E,Fernandez M,Salvador-Temprano E,et al.A reliable enzyme linked immunosorbent assay for African swine fever using the major structural protein as antigenic reagent[J].Archives of virology,1981,70(3):297-300.
[24]Gallardo C,Blanco E,Rodríguez J M,et al.Antigenic properties and diagnostic potential of African swine fever virus protein pp62expressed in insect cells[J].Journal of clinical microbiology,2006,44(3):950-956.
[25]Tulman E R,Rock D L.Novel virulence and host range genes of African swine fever virus[J].Current opinion in microbiology,2001,4(4):456-461.
[26]Perez-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 p30 protein produced in Trichoplusia ni larvae[J].Journal of Clinical Microbiology,2006,44(9):3114-3121.
[27]储德文.非洲猪瘟病毒p30重组蛋白的表达及单克隆抗体制备[D].扬州:扬州大学,2013.