非洲猪瘟病毒强免疫原性重组CD2v抗原的制备与初步应用

doi:10.11843/j.issn.0366-6964.2020.10.015

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (10): 2472-2480.doi: 10.11843/j.issn.0366-6964.2020.10.015

非洲猪瘟病毒强免疫原性重组CD2v抗原的制备与初步应用

周晓慧¹, 肖景景¹, 张鑫宇¹, 张泉¹, 夏晓莉¹, 孙怀昌^1,2*

1. 扬州大学兽医学院, 江苏高校动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009;
2. 江苏农牧职业技术学院, 泰州 225300

收稿日期:2020-02-20 出版日期:2020-10-25 发布日期:2020-10-26
通讯作者: 孙怀昌,主要从事预防兽医学研究,E-mail:sunh@yzu.edu.cn
作者简介:周晓慧(1993-),女,安徽亳州人,博士生,主要从事动物疫苗研究,E-mail:89507732@qq.com
基金资助:
国家重点研发计划项目（2017YFD0502303；2018YFC0840404-3）；江苏高校优势学科建设工程资助项目（PAPD）

Preparation and Preliminary Application of Highly Immunogenic Recombinant CD2v Antigen of African Swine Fever Virus

ZHOU Xiaohui¹, XIAO Jingjing¹, ZHANG Xinyu¹, ZHANG Quan¹, XIA Xiaoli¹, SUN Huaichang^1,2*

1. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China;
2. The Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Jiangsu Agri-Animal Husbandry Vocational College, Taizhou 225300, China

Received:2020-02-20 Online:2020-10-25 Published:2020-10-26

摘要/Abstract

摘要： 旨在获得非洲猪瘟病毒（ASFV）强免疫原性重组CD2v抗原，利用生物信息学软件进行CD2v抗原指数分析，将其细胞质内免疫显性区与类弹性蛋白多肽（ELP）在重组大肠杆菌中进行融合表达，对ELP-CD2v融合蛋白的相变循环（ITC）条件进行优化，在优化条件下进行融合蛋白纯化，利用烟草蚀纹病毒（TEV）蛋白酶活性包涵体切除ELP标签，通过免疫转印法对重组CD2v抗原进行鉴定，利用重组CD2v抗原建立ELISA抗体检测方法，与多抗原ELISA对ASFV抗体阳性和阴性血清进行平行检测。结果显示，ELP-CD2v融合蛋白获得正确、可溶性表达，ITC条件为28℃和1.5 mol·L^-1 NaCl，在0.2% Triton X-100存在下进行ITC，纯化的融合蛋白纯度为76.3%；TEV蛋白酶活性包涵体能有效切割ELP标签，再次ITC回收的重组CD2v抗原纯度为91.7%，能被ASFV抗体识别；根据多抗原ELISA检测结果选择血清样品，用重组CD2v抗原ELISA进行检测，结果显示，15份ASFV抗体阴性血清均为CD2v抗体检测阴性，15份ASFV抗体阳性血清均为CD2v抗体检测阳性。这些研究结果表明，ASFV的CD2v蛋白胞内区存在强免疫原性表位，其重组抗原有望用于CD2v的抗体检测。

关键词: 非洲猪瘟病毒, CD2v蛋白, 强免疫原性抗原, 初步应用

Abstract: To obtain the highly immunogenic recombinant CD2v antigen of African swine fever virus (ASFV), the amino acid sequence of CD2v was analyzed for antigenic index using bioinformatics software and the intracytoplasmic region with high antigenic index was expressed in E. coli as an elastin-like polypeptide (ELP) fusion protein. After optimization of the conditions for inverse transition cycling (ITC), ELP-CD2v fusion protein was purified by ITC in the presence of different concentrations of Triton X-100 and the ELP tag was cleaved with active inclusion bodies of tobacco etch virus (TEV) protease. The tag-free recombinant CD2v antigen was recovered by an additional round of ITC and identified by Western blotting. By using the recombinant CD2v antigen, an indirect ELISA was established and used to detect ASFV antibody-positive and antibody-negative sera in parallel with the multi-antigen ELISA. The results showed that ELP-CD2v fusion protein was expressed correctly in E. coli with an optimal transition temperature of 28 ℃ at 1.5 mol·L^-1 NaCl. After one cycle of ITC in the presence of 0.2% Triton X-100, ELP-CD2v fusion protein was purified to 76.3% purity. The ELP tag was cleaved efficiently with the TEV protease and removed after an additional round of ITC. The recovered recombinant CD2v protein had a purity of 91.7%, which was recognized by pig anti-ASFV serum. For 30 serum samples detected by ASFV multi-antigen ELISA, recombinant CD2v ELISA showed that all of 15 antibody-negative sera were CD2v antibody negative and all of 15 antibody-positive sera were CD2v antibody positive. These data suggest that the presence of immune dominant epitopes in the intracytoplasmic region of CD2v protein and the potential application of the recombinant CD2v antigen for ASFV antibody detection.

Key words: African swine fever virus, CD2v protein, highly immunogenic antigen, preliminary application

中图分类号:

S852.659.1

周晓慧, 肖景景, 张鑫宇, 张泉, 夏晓莉, 孙怀昌. 非洲猪瘟病毒强免疫原性重组CD2v抗原的制备与初步应用[J]. 畜牧兽医学报, 2020, 51(10): 2472-2480.

ZHOU Xiaohui, XIAO Jingjing, ZHANG Xinyu, ZHANG Quan, XIA Xiaoli, SUN Huaichang. Preparation and Preliminary Application of Highly Immunogenic Recombinant CD2v Antigen of African Swine Fever Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(10): 2472-2480.

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非洲猪瘟病毒强免疫原性重组CD2v抗原的制备与初步应用

Preparation and Preliminary Application of Highly Immunogenic Recombinant CD2v Antigen of African Swine Fever Virus

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