犬细小病毒VP2基因克隆及原核表达分析

doi:10.16431/j.cnki.1671-7236.2016.04.004

摘要/Abstract

摘要： 试验旨在了解河北与甘肃地区犬细小病毒流行情况,并通过原核表达系统获得犬细小病毒VP2蛋白,制备该蛋白的多克隆抗体,为进一步研究犬细小病毒致病机理和治疗方法奠定基础。从10份疑似感染犬细小病毒犬的病料中提取病毒基因组DNA,并以其作为模板进行VP2基因的PCR扩增,将目的片段进行测序和分析后克隆至原核表达载体pET-30a中,阳性质粒转化至大肠杆菌BL21(DE3)感受态细胞并诱导表达,诱导产物经SDS-PAGE鉴定表达后切割目的蛋白条带免疫豚鼠制备多克隆抗体。序列分析结果显示成功克隆VP2基因,10株分离株中CPV-2a型占80%,CPV-2b型占20%。本试验检测的毒株与部分中国毒株形成了一个分支,与韩国、美国分离株呈现一定差异,与意大利分离株同源性较低。Western blotting分析证实了通过原核表达系统成功获得的大小为70 ku左右的重组蛋白具有反应原性和免疫原性。该试验初步表明河北与甘肃地区以CPV-2a型为主,原核表达的VP2蛋白具有良好的抗原性,本试验结果为犬细小病毒的防控提供科学依据。

关键词: 犬细小病毒; VP2; 基因型; 原核表达

Abstract: The study was aimed to investigate the epidemic situation of canine parvovirus in Hebei and Gansu provinces.VP2 protein was expressed by prokaryotic system and polyclonal antibodies were prepared, which provided a basis for the further researches on pathogenesis and therapy of canine parvovirus.Virus genomic DNA was extracted from 10 epidemic materials of suspected infected dogs and VP2 gene was amplified.The target fragments of VP2 gene were cloned to pET-30a vector after sequencing and analysis, then the positive expression plasmids were transformed into E.coli BL21 (DE3) cell.Polyclonal antibodies were prepared by immunizing guinea pigs with the SDS-PAGE identification of purified protein.The genetic analysis results showed that the VP2 gene was successfully cloned and 80% samples were belonged to CPV-2a and 20% samples were belonged to CPV-2b.The isolated strains of the test and part of China strains were gathered into a branch which had certain distance with strains of Korea and USA, and that had low homology with strains of Italy.SDS-PAGE results indicated that the recombinant protein with a molecular weight of 70 ku was expressed by prokaryotic system.Western blotting results showed that the recombinant protein had good immuneoreactivity and immunogenicity.The study indicated that CPV-2a was the predominant gene type in Hebei and Gansu provinces, meanwhile VP2 protein expressed by prokaryotic system had good antigenicity.This research provided a basis for the future study of the prevention and control of canine parvovirus.

Key words: canine parvovirus; VP2; gene type; prokaryotic expression

中图分类号:

Q785

卫巧林, 张韵, 甄士伟, 殷相平. 犬细小病毒VP2基因克隆及原核表达分析[J]. , 2016, 43(4): 870-878.

WEI Qiao-lin, ZHANG Yun, ZHEN Shi-wei, YIN Xiang-ping. Analysis of Clone and Prokaryotic Expression of Canine Parvovirus VP2 Gene[J]. , 2016, 43(4): 870-878.

参考文献

[1] Pollock R V.Experimental canine parvovirus infection in dogs[J].Cornell Vet,1982,72(2):103-119.
[2] Carmichael L E,Binn L N.New enteric viruses in the dog[J].Adv Vet Sci Comp Med,1981,25:31-37.
[3] Kelly W R.An enteric disease of dogs reselmbing feline panleucopaenia[J].Aust Vet J,1978,54(12):593.
[4] Reed A P,Jones E V,Miller T J.Nucleotide sequence and genome organization of canine parvovirus[J].J Virol,1988,62(1):266-276.
[5] López de Turiso J A,Cortés E,Ranz A,et al.Fine mapping of canine parvovirus B cell epitopes[J].J Gen Virol,1991,72(12):2445-2456.
[6] 杨玲,徐向明,殷俊,等.犬细小病毒分离株VP2基因的克隆与序列分析[J].扬州大学学报,2002,2(3):12-13.
[7] Langeveld J P,Casal J I,Vela C,et al.B-cell epitopes of canine parvovirus:Distribution on the primary structure and exposure on the viral surface[J].J Virol,1993,67(2):765-772.
[8] Truyen U.Evolution of canine parvovirus——A need for new vaccines?[J].Vet Microbiol,2006,117(1):9-13.
[9] Truyen U.Emergence and recent evolution of canine parvovirus[J].Vet Microbiol,1999,69(1-2):47-50.
[10] Parrish C R,Aquadro C F,Strassheim M L,et al.Rapid antigenic-type replacement and DNA sequence evolution of canine parvovirus[J].J Virol,1991,65(12):6544-6552.
[11] Decaro N,Elia G,Campolo M,et al.New approaches for the molecular characterization of canine parvovirus type 2 strains[J].J Vet Med B Infect Dis Vet Public Health,2005,52(7-8):316-319.
[12] Buonavoglia C,Martella V,Pratelli A,et al.Evidence for evolution of canine parvovirus type 2 in Italy[J].J Gen Virol,2001,82(12):3021-3025.
[13] Soma T,Taharaguchi S,Ohinata T,et al.Analysis of the VP2 protein gene of canine parvovirus strains from affected dogs in Japan[J].Res Vet Sci,2013,94(2):368-371.
[14] 叶俊华,马长书,徐玉生,等.犬细小病毒病血清学调查[J].中国养犬杂志,1997,8(1):7-9.
[15] Zhao Y B, Lin Y, Zeng X J,et al.Genotyping and pathobiologic characterization of canine parvovirus circulating in Nanjing,China[J].Virology Journal,2013,10:272-281.
[16] Nandi S,Chidri S,Kumar M,et al.Occurrence of canine parvovirus type 2c in the dogs with haemorrhagic enteritis in India[J].Res Vet Sci,2010,88:169-171.
[17] Kang B K,Song D S,Lee C S,et al.Prevalence and genetic characterization of canine parvoviruses in Korea[J].Virus Genes,2008,36:127-133.
[18] Ohshima T,Hisaka M,Kawakami K,et al.Chronological analysis of canine parvovirus type 2 isolates in Japan[J].J Vet Med Sci,2008,70(8):769-775.
[19] Zhang R,Yang S,Zhang W,et al.Phylogenetic analysis of the VP2 gene of canine parvoviruses circulating in China[J].Virus Genes,2010,40:397-402.
[20] 杨晓农,张焕容,杨发龙,等.成都地区犬细小病毒VP2基因克隆分析及基因型鉴定[J].中国畜牧兽医,2011,38(9):105-110.
[21] 许冬蕾,任常宝,张晓战,等.广州地区犬细小病毒VP2基因的序列分析[J].中国畜牧兽医,2013,40(12):38-42.
[22] Zhang Q,Xu X M,Zhai G Q,et al.Molecular characterisation of canine parvovirus strains circulating in China[J].Afr J Biotechnol,2010,9:4556-4560.
[23] Xu J,Guo H C,Wei Y Q,et al.Phylogenetic analysis of canine parvovirus isolates from Sichuan and Gansu provinces of China in 2011[J].Transbound Emerg Dis,2015,62(1):91-95.
[24] Pereira C A,Leal E S,Durigon E L.Selective regimen shift and demographic growth increase associated with the emergence of high-fitness variants of canine parvovirus[J].Infect,Genet Evol,2007,7:399-409.
[25] Han S C,Guo H C,Sun S Q,et al.Full-length genomic characterizations of two canine parvoviruses prevalent in Northwest China[J].Arch Microbiol,2015,197:621-626.
[26] Yi L,Tong M,Cheng Y,et al.Phylogenetic analysis of canine parvovirus VP2 gene in China[J]. Transbound Emerg Dis,2016,63(2):e262-e269.
[27] Zhong Z,Liang L,Zhao J,et al.First isolation of new canine parvovirus 2a from Tibetan mastiff and global analysis of the full-length VP2 gene of canine parvoviruses 2 in China[J].Int J Mol Sci,2014,15:12166-12187.
[28] Hueffer K,Parker J S,Weichert W S,et al.The natural host range shift and subsequent evolution of canine parvovirus resulted from virus-specific binding to the canine transferrin receptor[J].J Virol,2003,77:1718-1726.
[29] Jeoung S Y,Ahn S J,Kim D.Genetic analysis of VP2 gene of canine parvovirus isolates in Korea[J].Vet Med Sci,2008,70:719-722.
[30] Phromnoi S,Sirinarumitr K,Sirinarumitr T.Sequence analysis of VP2 gene of canine parvovirus isolates in Thailand[J].Virus Genes,2010,41:23-29.
[31] Csagola A,Varga S,Lorincz M,et al.Analysis of the full-length VP2 protein of canine parvoviruses circulating in Hungary[J].Arch Virol,2014,159(9):2441-2444.
[32] Geng Y,Guo D,Li C,et al.Co-circulation of the rare CPV-2c with unique Gln370Arg substitution, new CPV-2b with unique Thr440Ala substitution,and new CPV-2a with high prevalence and variation in Heilongjiang province,northeast China[J].PLoS One,2015,10(9):e0137288.