[非洲猪瘟病毒在重组蛋白CD2v、pX69R和pE248R存在情况下的复制特征。]

[ASF virus replication features in the presence of recombinant proteins CD2v, pX69R and pE248R.].

作者信息

Mazloum A, Zhukov I U, Aronova E B, Igolkin A S, Vlasova N N

机构信息

FGBI «ARRIAH» Federal State Budgetary Institution «Federal Center for Animal Health» Vladimir region, Vladimir city, Yuryevets microdistrict, 600901, Russian Federation.

出版信息

Vopr Virusol. 2019;64(4):193-200. doi: 10.36233/0507-4088-2019-64-4-193-200.

DOI:10.36233/0507-4088-2019-64-4-193-200

PMID:32163686

Abstract

INTRODUCTION

African swine fever (ASF), sever hemorrhagic disease of swine caused by a large DNA virus of the Asfaviridae family. Since there are no effective and safe vaccines against ASF yet, it is urgent to study the functions of its proteins, which is applicable by analyzing the features of ASF virus replication in the presence of recombinant proteins in vitro.

PURPOSE

To study the effect of ASFV recombinant proteins CD2v, pE248R and pX69R on the speed and level of reproduction of ASF virus in vitro. Thus, obtain the necessary knowledge to develop approaches for creating a vaccine against ASF.

MATERIALS AND METHODS

ASFV isolate Krasnodar 07/17 and strain ASF/ARRIAH/CV-1 were used. Cloning of X69R, EP402R, and E248R genes was performed in the pJET1.2 / blunt vector and pCI-neo in E. coli JM-109 cells, according to the manufacturer's manual. Localization of recombinant proteins in CV-1 cell line carried out by direct immunofluorescence reaction (DIF) using polyclonal antibodies conjugated to FITC. The ASF virus reproduction level was assessed by hemadsorption reaction and qPCR kit (Central Research Institute of Epidemiology).

RESULTS

Recombinant plasmids pCI-neo / E248R, pCI-neo / EP402R and pCI-neo / X69R were constructed. The localization and the specificity of the obtained recombinant proteins CD2v, pE248R and pX69R was confirmed. It was established that these recombinant proteins induce the level of ASF virus reproduction on days 3-5 of the experiment by ~ 1.2-1.5 lgHADU/cm in comparison with the negative control.

DISCUSSION

The data obtained demonstrate the important role of CD2v, pX69R and pE248R proteins in the reproduction of the virus, since they significantly affect its level. The exact function of pX69R protein was not determined, however, in the experiments its positive effect on ASF virus reproduction was established, manifested in an increase in its reproduction level.

CONCLUSION

This methodology allows us to study the nature of the effect of proteins with unknown function on ASF virus replication.

摘要

引言

非洲猪瘟（ASF）是由非洲猪瘟病毒科的一种大型DNA病毒引起的猪的严重出血性疾病。由于目前尚无针对ASF的有效且安全的疫苗，因此迫切需要研究其蛋白质的功能，这可通过在体外重组蛋白存在的情况下分析ASF病毒复制的特征来实现。

目的

研究非洲猪瘟病毒（ASFV）重组蛋白CD2v、pE248R和pX69R对ASF病毒体外繁殖速度和水平的影响。从而获得开发ASF疫苗方法所需的知识。

材料与方法

使用ASFV分离株克拉斯诺达尔07/17和ASF/ARRIAH/CV-1毒株。按照制造商的说明书，在大肠杆菌JM-109细胞中，将X69R、EP402R和E248R基因克隆到pJET1.2/ blunt载体和pCI-neo中。使用与异硫氰酸荧光素（FITC）偶联的多克隆抗体，通过直接免疫荧光反应（DIF）在CV-1细胞系中定位重组蛋白。通过血细胞吸附反应和qPCR试剂盒（中央流行病学研究所）评估ASF病毒的繁殖水平。

结果

构建了重组质粒pCI-neo/E248R、pCI-neo/EP402R和pCI-neo/X69R。证实了所获得的重组蛋白CD2v、pE248R和pX69R的定位和特异性。结果表明，与阴性对照相比，在实验的第3至5天，这些重组蛋白使ASF病毒的繁殖水平提高了约1.2 - 1.5 lgHADU/cm²。