兔出血症病毒镇江株的分离鉴定及VP60蛋白活性表达

doi:10.16431/j.cnki.1671-7236.2018.12.028

摘要/Abstract

摘要：

试验旨在分离兔出血症病毒（rabbit hemorrhagic disease virus，RHDV）镇江株，分析其遗传进化变异，并表达具有良好活性的重组VP60蛋白。通过排除细菌感染、血凝（HA）和血凝抑制（HI）检测、动物攻毒试验与病毒传代、LD₅₀测定等方法，自江苏省镇江市某兔场发病死亡动物肝脏组织样品中分离病原并鉴定；RT-PCR方法获得VP60基因，通过分析VP60基因核苷酸及氨基酸序列研究其遗传进化；将VP60基因与pCold-Sumo载体连接，构建低温诱导、融合Sumo标签原核表达载体，15℃、IPTG诱导表达重组VP60蛋白并对表达产物进行反应原性鉴定。结果显示，分离鉴定获得RHDV ZJ2015毒株，该毒株能凝集人"O"型红血球，HA效价为11log2，其血凝性能被RHDV （AV33）抗血清抑制，该毒株的LD₅₀为10^-6.38/mL，具有较强的毒力；RT-PCR扩增得到大小约为1 740 bp的特异性条带，系统进化树分析显示，该毒株属于RHDV1抗原遗传变异株（RHDVa），与RHDV1和RHDV2 VP60基因核苷酸序列同源性分别为89.4%~97.6%和81.1%~81.5%，氨基酸序列同源性分别为93.8%~98.3%和87.4%~87.6%。构建的低温原核融合表达质粒pCold-VP60在大肠杆菌Rosetta （DE3）中成功表达，通过SDS-PAGE及Western blotting分析，在分子质量74 ku处有特异性表达蛋白条带，且与抗血清发生特异性抗原抗体结合反应，说明重组蛋白具有良好的反应原性。本研究为开展兔病毒性出血症流行病学研究、开发新型重组疫苗与诊断试剂提供参考依据。

关键词: 兔出血症病毒(RHDV); 分离鉴定; 遗传进化; 低温诱导表达; 活性

Abstract:

The aim of this study was to isolate the rabbit hemorrhagic disease virus (RHDV) strain from Zhenjiang and analyze genetic evolution and variation,as well as prokaryotic expression recombinant VP60 protein with good reactionogenicity.By means of excluding bacterial infection,hemagglutination (HA) and hemagglutination inhibition (HI) test,animal challenge test and virus passage,LD₅₀ determination,the pathogen was isolated and identified from the liver tissue sample of infected dead animals in a rabbit farm located in Zhenjiang city,Jiangsu province.The virus VP60 gene was obtained by RT-PCR,and the nucleotide and amino acid sequences were analyzed to study its genetic evolution.The low temperature inducible and Sumo-tag fused prokaryotic expression vector was constructed via recombining the VP60 gene into a low temperature induction vector pCold-Sumo,and the recombinant protein was expressed induced with IPTG at 15℃ overnight,and the reactivity was identified thereafter.The results showed that the RHDV ZJ2015 strain was isolated,and the virus could strongly agglutinate human type O erythrocytes,the HA titer reached to 11log2,and this activity could be blocked by the RHDV (AV33) antiserum.The LD₅₀ of this strain reached to 10^-6.38/mL,indicating the virus was highly lethal to rabbit.The size of the specific PCR product was about 1 740 bp,and the strain was classified as the antigen genetic variant (RHDVa) of RHDV1 according to the phylogenetic tree based on the nucleotide sequences.The nucleotide sequence homologies compared to RHDV1 and RHDV2 were 89.4% to 97.6% and 81.1% to 81.5%,and the amino acid sequence homologies were 93.8% to 98.3% and 87.4% to 87.6%,respectively.The recombinant VP60 fused with the Sumo-tag was successfully expressed in E.coli Rosetta (DE3) cells,the molecular weight was about 74 ku via SDS-PAGE,and the recombinant protein had a good reactivity with anti-serum via Western blotting analysis.This study laid the foundation for the epidemiological study of rabbit hemorrhagic disease and the development of recombinant vaccines and diagnostic reagents.

Key words: rabbit hemorrhagic disease virus (RHDV); isolation and identification; genetic evolution; low temperature induced expression; activity

中图分类号:

S852.65

杨慧, 于雪, 孙培娇, 王艳, 刘吉山, 王玉茂, 沈志强, 肖跃强. 兔出血症病毒镇江株的分离鉴定及VP60蛋白活性表达[J]. 《中国畜牧兽医》, 2018, 45(12): 3545-3554.

YANG Hui, YU Xue, SUN Peijiao, WANG Yan, LIU Jishan, WANG Yumao, SHEN Zhiqiang, XIAO Yueqiang. Isolation,Identification of a Strain of Zhenjiang RHDV and Active Expression of VP60 Protein[J]. , 2018, 45(12): 3545-3554.

参考文献

[1] 刘胜江,薛华平,浦伯清,等.兔的一种新病毒病——兔病毒性出血症[J].畜牧与兽医,1984,16(6):253-255. LIU S J,XUE H P,PU B Q,et al.A new viral disease in rabbits——Rabbit hemorrhagic disease[J].Animal Husbandry & Veterinary Medicine,1984,16(6):253-254.(in Chinese)
[2] ABRANTES J,VAN DER LOO W,LE PENDU J,et al.Rabbit haemorrhagic disease (RHD) and rabbit haemorrhagic disease virus (RHDV):A review[J].Veterinary Research,2012,43(1):12.
[3] 刘光清,张玉颖,云涛,等.兔病毒性出血症病毒基因组3'端非编码区的分子克隆及生物信息学分析[J].西北农林科技大学学报(自然科学版),2006,34(3):16-20. LIU G Q,ZHANG Y Y,YUN T,et al.Cloning and analyzing of the secondary structure of the 3' noncoding region of RHDV[J]. Journal of Northwest A&F University (Nature Science Edition),2006,34(3):16-20.(in Chinese)
[4] 祖立闯,沈志强,李娇,等.兔病毒性出血症病毒VP60蛋白在诊断与亚单位疫苗应用中的研究进展[J].畜牧与兽医,2012,44(9):100-103. ZU L C,SHEN Z Q,LI J.Advances in research of rabbit viral hemorrhagic disease virus VP60 protein in diagnosis and subunit vaccine application[J].Animal Husbandry & Veterinary Medicine,2012,44(9):100-103.(in Chinese)
[5] ISOMURSU M,NEIMANIS A,KARKAMO V,et al.An outbreak of rabbit hemorrhagic disease in Finland[J].Journal of Wildlife Diseases,2018,50(4):1-5.
[6] ROUCO C,ABRANTES J,SERRONHA A,et al.Epidemiology of RHDV2(Lagovirus europaeus/GI.2) in free-living wild European rabbits in Portugal[J].Transboundary and Emerging Diseases,2018,65(2):e373-e382.
[7] NEIMANIS A S,AHOLA H,ZOHARI S,et al.Arrival of rabbit haemorrhagic disease virus 2 to Northern Europe:Emergence and outbreaks in wild and domestic rabbits (Oryctolagus cuniculus) in Sweden[J].Transboundary and Emerging Diseases,2018,65(1):213-220.
[8] LE GALL-RECULE G,LEMAITRE E,BERTAGNOLI S,et al.Large-scale lagovirus disease outbreaks in European brown hares (Lepus europaeus) in France caused by RHDV2 strains spatially shared with rabbits (Oryctolagus cuniculus)[J].Veterinary Research,2017,48(1):70.
[9] LE GALL-RECULE G,LAVAZZA A,MARCHANDEAU S,et al.Emergence of a new lagovirus related to rabbit haemorrhagic disease virus[J].Veterinary Research,2013,44:81.
[10] NEIMANIS A,LARSSON PETTERSSON U,HUANG N,et al.Elucidation of the pathology and tissue distribution of Lagovirus europaeus GI.2/RHDV2(rabbit haemorrhagic disease virus 2) in young and adult rabbits (Oryctolagus cuniculus)[J].Veterinary Research,2018,49(1):46.
[11] PEACOCK D,KOVALISKI J,SINCLAIR R,et al.RHDV2 overcoming RHDV immunity in wild rabbits (Oryctolagus cuniculus) in Australia[J].The Veterinary Record,2017,180(11):280.
[12] CARVALHO C L,DUARTE E L,MONTEIRO M,et al.Challenges in the rabbit haemorrhagic disease 2(RHDV2) molecular diagnosis of vaccinated rabbits[J].Veterinary Microbiology,2017,198:43-50.
[13] FORRESTER N L,TROUT R C,TURNER S L,et al.Unravelling the paradox of rabbit haemorrhagic disease virus emergence,using phylogenetic analysis;Possible implications for rabbit conservation strate-gies[J].Biological Conservation,2006,131(2):296-306.
[14] XU Z J,CHEN W X.Viral haemorrhagic disease in rabbits:A review[J].Veterinary Research Communications,1989,13(3):205-212.
[15] CAPUCCI L,FALLACARA F,GRAZIOLI S,et al.A further step in the evolution of rabbit hemorrhagic disease virus:The appearance of the first consistent antigenic variant[J].Virus Research,1998,58(1-2):115-126.
[16] 田浪,王红宁,李建文,等.一株无血凝性兔出血症病毒的分离鉴定及其衣壳蛋白VP60基因的克隆和序列分析[J].畜牧兽医学报,2006,37(10):1078-1083. TIAN L,WANG H N,LI J W,et al.Isolation and identification of a non-haemagglutinating strain of RHDV from China and cloning and sequence analysis of VP60 gene of the strain[J].Acta Veterinaria et Zootechnica Sinica,2006,37(10):1078-1083.(in Chinese)
[17] 肖跃强.兔出血症病毒遗传变异分析及抗原抗体检测方法的建立与应用[D].长春:吉林大学,2014. XIAO Y Q.Genetic variation analysis of RHDV and establishment and application of detecting methods for viruses and antibodies[D].Changchun:Jilin University,2014.(in Chinese)
[18] JIANG Q,YU Z,LIU J S,et al.Recombinant canine adenovirus type 2 expressing rabbit hemorrhagic disease virus VP60 protein provided protection against RHD in rabbits[J].Veterinary Microbiology,2018,213:15-20.
[19] ZHENG X,WANG S,ZHANG W,et al.Development of a VLP-based vaccine in silkworm pupae against rabbit hemorrhagic disease virus[J].International Immunopharmacology,2016,40:164-169.
[20] GUO H,ZHU J,TAN Y,et al.Self-assembly of virus-like particles of rabbit hemorrhagic disease virus capsid protein expressed in Escherichia coli and their immunogenicity in rabbits[J].Antiviral Research,2016,131:85-91.
[21] YANG D K,KIM H H,NAH J J,et al.Rabbit hemorrhagic disease virus variant recombinant VP60 protein induces protective immunogenicity[J].Journal of Microbiology and Biotechnology,2015,25(11):1960-1965.
[22] LOPEZ-VIDAL J,GOMEZ-SEBASTIAN S,BARCENA J,et al.Improved production efficiency of virus-like particles by the baculovirus expression vector system[J].PLoS One,2015,10(10):e0140039.
[23] SPIBEY N,MCCABE V J,GREENWOOD N M,et al.Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease[J].Veterinary Record,2012,170(12):309.
[24] 李传山,任力刚,李维英,等.兔出血症病毒YL株衣壳蛋白VP60基因在原核系统中的高效表达[J].西北农林科技大学学报(自然科学版),2007,35(11):24-28. LI C S,REN L G,LI W Y,et al.High expression of capsid protein (VP60) gene from rabbit hemorrhagic disease virus YL strain in E.coli[J].Journal of Northwest A&F University (Nature Science Edition),2007,35(11):24-28.(in Chinese)
[25] 隋慧.兔出血症病毒主要结构蛋白基因的克隆、原核表达及其免疫原性鉴定[J].中国畜牧兽医,2012,39(5):76-78. SUI H.Cloning,expression and immunogenicity of major structural protein gene of rabbit hemorrhagic disease virus[J].China Animal Husbandry & Veterinary Medicine,2012,39(5):76-78.(in Chinese)
[26] 王永山,陆承平,周宗安.兔出血症病毒NJ85株衣壳蛋白基因在原核系统中的高效表达[J].农业生物技术学报,2004,12(4):408-411. WANG Y S,LU C P,ZHOU Z A.High expression of the capsid protein gene of the Chinese early isolate NJ85 of rabbit hemorrhagic disease virus in Escherichia coli[J].Journal of Agricultural Biotechnology,2004,12(4):408-411.(in Chinese)
[27] BOGA J A,CASAIS R,MARIN M S,et al.Molecular cloning,sequencing and expression in Escherichia coli of the capsid protein gene from rabbit haemorrhagicdisease virus (Spanish isolate AST/89)[J].The Journal of General Virology,1994,75(Pt 9):2409-2413.