非洲猪瘟病毒CD2v胞内区蛋白间接ELISA方法的建立及初步应用

doi:10.3969/j.issn.1005-944X.2023.03.017

首页 > 过刊浏览>2023年第40卷第3期 >97-104. DOI:10.3969/j.issn.1005-944X.2023.03.017

非洲猪瘟病毒CD2v胞内区蛋白间接ELISA方法的建立及初步应用
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                        10.3969/j.issn.1005-944X.2023.03.017
                    
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中图分类号:S852.65
基金项目:广东省科技计划项目（2021B1212030015）；广东省重点领域研发计划资助项目（2019B2021003）

Establishment and Preliminary Application of an Indirect ELISA for CD2v Intracellular Protein of African Swine Fever Virus

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摘要:

近年来，在全球范围内出现了EP402R基因（编码CD2v蛋白）缺失的ASFV突变毒株，因此需要开发一种检测方法来区分CD2v缺失的自然突变株和野生型ASFV毒株。对ASFV国内分离株WUHAN 2019-1株的EP402R基因序列，利用生物信息学分析其亲水性，选取可溶性较强部分（232~333 aa），根据大肠杆菌密码子偏嗜性对该段基因进行优化并人工合成，插入原核表达载体pET-28a进行低温表达，获得带有HIS标签的可溶性CD2v胞内区重组蛋白，将其命名为pET-28a-CD2v-IS，大小约15 ku。Western-blot鉴定结果显示，该重组蛋白抗原性良好。利用纯化后的CD2v重组蛋白建立了间接ELISA方法。经反应条件优化，最终确定最佳包被质量浓度为 0.5 μg/mL，封闭时间为1 h，样本最佳稀释度为1:10，样本最佳孵育时间为1 h，二抗最佳稀释度为1:20 000，最佳孵育时间为45 min，底物孵育最佳时间为5 min；确定建立的间接ELISA方法的S/N临界判定值为 1.61。试验灵敏性可达1:1 280；批内变异系数小于4.15%，批间变异系数小于9.84%；只与ASFV全病毒株阳性血清发生反应，与ASFV（CD2v）缺失株、猪瘟病毒（CSFV）、猪繁殖与呼吸综合征病毒（PRRSV）等其他病毒阳性血清均无交叉反应；在检测46 份背景清晰与54份背景未知的血清时，与商用试剂盒符合率分别达到 100%与 90.7%。结果表明，本研究建立的间接ELISA方法具有良好的特异性、敏感性与重复性，检测结果与商品化试剂盒和WOAH推荐的间接ELISA方法一致，如果结合其他ASFV结构蛋白制成的ELISA检测试剂盒，可区分ASFV野毒与CD2v缺失株感染。

Abstract:

In recent years，ASFV mutant strains with deletion of EP402R gene（encoding CD2v protein）appeared all over the world，which required a tool to distinguish them from the wild ones. For the EP402R gene sequence of WUHAN 2019-1 strain，a domestic isolate of ASFV，its hydrophily was analyzed using bioinformatics to select the segments with high solubility（232~333 aa），which were optimized and artificially synthesized according to the codon bias of Escherichia coli. The soluble CD2v intracellular recombinant protein with HIS tag，about 15 ku and named as pET-28a-CD2v-IS，was obtained through inserting the prokaryotic expression vector pET-28a to express it at a low temperature. Based on the results by Western-blot，it was found that the recombinant protein was with good antigenicity. An indirect ELISA was established by using the purified CD2v recombinant protein. After optimizing the reaction conditions，the optimal coating concentration was determined as 0.5 μg/mL with the sealing time of one hour. The optimal dilution concentration of samples was 1:10 with the optimal incubation time of one hour，the optimal dilution concentration of secondary antibody was 1:20 000 with the optimal incubation time of 45 min. The optimal incubation time of substrate was 5 min；the S/N critical value of the established indirect ELISA was 1.61. The test sensitivity could reach 1:1 280；the intra-assay coefficient of variation（CV）was less than 4.15%，while the inter-assay one was less than 9.48%；the established method only reacted with the positive sera of ASFV whole virus，but failed with the positive sera of ASFV（CD2v）deletion strain，classical swine fever virus（CSFV），porcine reproductive and respiratory syndrome virus（PRRSV）and other viruses；and its coincidence rates with commercial kits were 100% and 90.7%，respectively，when 46 sera with clear background and 56 with unknown background were tested. In conclusion，the method established in the study was with good specificity，sensitivity and repeatability，and its results were consistent with those by commercial kits and the indirect ELISA proposed by WOAH，ASFV wild virus could be distinguished from CD2v detection strain with the help of ELISA kits made of other ASFV structural proteins.