Recombinant Protein p30 for Serological Diagnosis of African Swine Fever by Immunoblotting Assay
Recombinant Protein p30 for Serological Diagnosis of African Swine Fever by Immunoblotting Assay
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DOI：
10.1111/tbed.12539
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发表时间：
2017-10-01
影响因子：
4.3
通讯作者：
Sereda, A. D.
中科院分区：
文献类型：
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作者：
Kazakova, A. S.;Imatdinov, I. R.;Sereda, A. D.
本文致力于开发和评价用于非洲猪瘟（ASF）血清学诊断的免疫印迹试验系统，该系统基于目前在俄罗斯联邦流行的ASF病毒（ASFV）代表株斯塔夫罗波尔01/08（斯塔夫罗波尔2008）的高度纯化重组p30。本研究的主要工作包括：（i）将ASFV基因CP 204 L（p30）的中心亲水区克隆到原核表达载体中;（ii）表达并纯化含有硫氧还蛋白和多聚组氨酸位点的重组p30（p30e1_TrxA_6xHis）;（iii）免疫印迹试验系统的开发（iv）使用来自实验性或自然感染ASFV的家猪和野猪的血清和器官样品评估Rec p30-IB。Rec p30-IB的测试显示，该测定的诊断特异性和灵敏度分别为98.75%和100.00%。Rec p30-IB的高灵敏度允许从感染后6至8天开始检测从家猪和野猪收集的免疫系统器官和血清样品中的ASFV特异性抗体，而不管样品的病毒毒力、血清免疫型和地理来源（东欧、南欧、西欧、中非和东南非）。
This article is devoted to the development and evaluation of the immunoblotting test system for serological diagnosis of African swine fever (ASF), based on the highly purified recombinant p30 of ASF virus (ASFV) strain Stavropol 01/08 (Stavropol 2008), representative of the ASFV currently circulating in the Russian Federation. The main project stages are as follows: (i) cloning of the central hydrophilic region of the ASFV gene CP204L (p30) into a prokaryotic vector; (ii) expression and chromatographic purification of the recombinant product p30 with thioredoxin and poly-histidine site (p30e1_TrxA_6xHis); (iii) development of the immunoblotting test system (Rec p30-IB) using the highly purified recombinant p30; and (iv) evaluation of Rec p30-IB using sera and organ samples from domestic pigs and wild boars experimentally or naturally infected by ASFV. Testing of the Rec p30-IB showed the diagnostic specificity and sensitivity of the assay to be 98.75% and 100.00%, respectively. High sensitivity of the Rec p30-IB allowed the detection of ASFV-specific antibodies in samples of organs of the immune system and blood sera, collected from domestic pigs and wild boars, starting from 6 to 8days post-infection, regardless of virus virulence, seroimmunotype and geographic origin of the samples (East Europe, South Europe, West Europe, Central and south-east Africa).