非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位

doi:10.3864/j.issn.0578-1752.2022.15.016

摘要/Abstract

摘要：

【目的】制备非洲猪瘟病毒（African swine fever virus,ASFV）p30蛋白的单克隆抗体（monoclonal antibodies,MAbs）并初步分析其所识别的线性抗原表位,为ASFV及其抗体检测方法的建立及p30蛋白结构和功能的研究奠定基础。【方法】将原核表达并纯化的p30重组蛋白作为免疫原,免疫6—8周龄BALB/c雌鼠,每两周免疫1次,共免疫3次,首次免疫是抗原与等体积的弗氏完全佐剂乳化后免疫,第二次和第三次免疫与等体积的弗氏不完全佐剂乳化,3次免疫后1 w断尾采血,间接酶联免疫吸附试验（ELISA）检测血清抗体效价,选择血清效价最高的小鼠进行加强免疫,3 d后取小鼠脾淋巴细胞与SP2/0骨髓瘤细胞按照4﹕1的比例使用PEG进行常规细胞融合。利用重组p30蛋白作为包被抗原,间接ELISA筛选阳性杂交瘤细胞,有限稀释法进行克隆纯化,直至筛出能够稳定分泌抗体的MAbs。将ASFV接种于猪肺泡巨噬细胞,以筛选的MAbs为一抗、兔抗鼠HRP-IgG为二抗,进行间接免疫荧光试验（IFA）。将感染和未感染ASFV的细胞沉淀处理后进行 SDS-PAGE并转印至硝酸纤维素膜,分别以IFA鉴定为阳性的MAbs上清为一抗、兔抗鼠HRP-IgG为二抗,进行Western blotting分析,筛选获得p30 MAbs。根据已知序列设计引物扩增p30ab与p30bc两段截短基因,其中p30ab代表由第86—153位氨基酸残基的截短体,p30bc代表由第120—187位氨基酸残基的截短体,原核表达部分重叠的截短p30蛋白,最终获得重组蛋白GST-p30ab与重组蛋白GST-p30bc。分别以GST-p30ab和GST-p30bc融合蛋白为包被抗原,以5株MAbs为一抗,以兔抗鼠HRP-IgG为二抗, 通过间接ELISA方法初步定位p30蛋白的抗原表位。【结果】以纯化的重组蛋白为包被抗原,经间接ELISA试验筛选出25株可分泌抗重组 p30蛋白的杂交瘤细胞株。IFA结果显示,5株MAbs（8F4、1D3、1H2、6C3和8E11）与ASFV感染的猪肺泡巨噬细胞IFA 试验呈阳性;Western blotting结果显示,5株MAbs均能够与ASFV感染的细胞呈阳性反应,与未感染病毒的细胞呈阴性反应。试验构建的p30截短体重组蛋白GST-p30ab以可溶和包涵体两种形式表达,而GST-p30bc仅以包涵体形式表达,以两组截短体融合蛋白为包被抗原,通过间接ELISA检测出MAbs 8F4、1H2和6C3与两个重组蛋白均能有效结合,证明MAbs 8F4、1H2和6C3抗原识别区域为两组截短蛋白重叠区域,即第120—153位氨基酸;MAbs 8E11与1D3则只能与GST-p30ab蛋白结合, 证明MAbs 8E11与1D3抗原识别区域为两个重组蛋白的非重叠区域,即第86—119位氨基酸。【结论】本研究可溶性地表达了p30蛋白的第86—153位氨基酸截短体重组蛋白,制备了5株p30 MAbs,定位到2个p30蛋白抗原表位。结合ELISA和IFA,可建立十分可靠的ASFV及其抗体的检测手段。

关键词: 非洲猪瘟病毒, p30蛋白, 酶联免疫吸附试验, 单克隆抗体, 抗原表位

Abstract:

【Objective】In this study, monoclonal antibodies (MAbs) against the p30 protein of African swine fever virus (ASFV) were prepared and the linear epitopes on p30 was analyzed, which could lay the foundation for ASFV and its antibody detection as well as the study of p30 protein structure and function.【Method】BALB/c female mice aged 6 to 8 weeks were immunized with prokaryotic expression and purified recombinant p30 protein. The mice were immunized once every two weeks, with three times in total. First immunization was done with emulsification of antigen and equal volume Freund’s complete adjuvant, then, the mice were immunized with emulsification of antigen and equal volume of Freund’s incomplete adjuvant for the second and third immunization. After three immunizations, the tail was cut off and the blood was collected, and the serum antibody titer was detected by indirect enzyme-linked immunosorbent assay (ELISA). The mice with the highest serum titer were selected for enhanced immunization. Three days later, the mice spleen lymphocytes and SP2/0 myeloma cells were fused with PEG at a ratio of 4﹕1. The positive hybridoma cells were screened by indirect ELISA by using the recombinant p30 protein as coated antigen. and the MAbs which could secrete antibodies steadily were cloned and purified by limited dilution method. ASFV was inoculated into porcine alveolar macrophages, and the indirect immunofluorescence assay (IFA) was performed with MAbs as primary antibody and rabbit anti-rat HRP-IgG as secondary antibody. The ASFV-infected and uninfected cells were precipitated and transferred to the nitrocellulose membrane by SDS-PAGE. p30 MAbs were obtained by Western blotting analysis of MAbs and identified positive by IFA, which was used as primary antibody and rabbit anti-rat HRP-IgG as secondary antibody. Primers were designed to amplify two truncated genes p30ab and p30bc, p30ab stands for truncated amino acid residues at position 86-153 and p30bc stands for truncated amino acid residues at position 120-153. The recombinant protein GST-p30ab and recombinant protein GST-p30bc were obtained by partial overlapping truncated p30 protein expression in prokaryotes. GST-p30ab and GST-p30bc fusion proteins were used as coated antigens, and the epitopes of p30 protein were preliminarily identified by indirect ELISA with 5 MAbs as primary antibodies, with rabbit anti-rat HRP-IgG as secondary antibodies. 【Result】The purified recombinant protein was used as the coated antigen, and 25 hybridoma cell lines were screened by indirect ELISA, which could secrete anti-recombinant p30 protein. IFA results showed that 5 MAbs (8F4, 1D3, 1H2, 6C3 and 8E11) were positive for ASFV-infected cells. Western blotting results showed that all 5 strains of MAbs could react positively with ASFV-infected cells and negatively with uninfected cells. The recombinant p30 protein GST-p30ab was expressed in soluble and inclusion body forms, and GST-p30bc was expressed in inclusion body form. The truncated fusion proteins of the two groups were used as the coated antigens. Indirect ELISA showed that MAbs 8F4, 1H2 and 6C3 could effectively bind to the two recombinant proteins that means MAbs 8F4, 1H2 and 6C3 recognized the epitope amino acid 120-153; MAbs 8E11 and 1D3 could only bind to GST-p30ab protein, which meant they recognized the epitope amino acid 86-119. 【Conclusion】In this study, the recombinant p30 protein with amino acid truncated at position 86-153 was expressed in soluble form. Five MAbs were prepared and two p30 protein epitopes were located. Combined with ELISA and IFA, it could be formed a very reliable method for the detection of ASFV and its antibody.

Key words: African swine fever virus, p30 protein, enzyme-linked immunosorbent assay, monoclonal antibody, epitope

魏天,王成宇,王凤杰,李忠鹏,张芳毓,张守峰,扈荣良,吕礼良,王永志. 非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位[J]. 中国农业科学, 2022, 55(15): 3062-3070.

WEI Tian,WANG ChengYu,WANG FengJie,LI ZhongPeng,ZHANG FangYu,ZHANG ShouFeng,HU RongLiang,LÜ LiLiang,WANG YongZhi. Preparation of Monoclonal Antibodies Against the p30 Protein of African Swine Fever Virus and Its Mapping of Linear Epitopes[J]. Scientia Agricultura Sinica, 2022, 55(15): 3062-3070.

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单克隆抗体 Monoclonal antibody	GST-p30ab	GST-p30bc	阴性对照Negative control	阳性对照 Positive control
8F4	2.223±0.05	2.131±0.09	0.101	0.531
1D3	2.129±0.07	0.122±0.002	0.072	0.483
8E11	2.053±0.05	0.093±0.002	0.058	0.392
6C3	2.276±0.04	2.025±0.09	0.079	0.516
1H2	2.173±0.04	1.958±0.07	0.068	0.481