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

Abstract

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

CLC Number:

S852.65

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.

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Isolation,Identification of a Strain of Zhenjiang RHDV and Active Expression of VP60 Protein

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