非洲猪瘟病毒p54蛋白单克隆抗体的制备及其抗原表位鉴定

doi:10.3864/j.issn.0578-1752.2024.19.015

摘要/Abstract

摘要：

【目的】获得可溶性非洲猪瘟病毒（African swine fever virus, ASFV）p54蛋白和抗p54蛋白的单克隆抗体（monoclonal antibodies, mAb），并分析其识别的线性抗原表位，为p54蛋白结构和功能的研究及血清学诊断试剂的研发奠定基础。【方法】构建了重组原核表达质粒pET-21a-E183L，将其转化至大肠杆菌BL21（DE3）感受态细胞中，表达并利用Ni2+柱和分子筛纯化得到p54重组蛋白。以该蛋白为抗原免疫5周龄SPF级BALB/c小鼠，每两周免疫一次，共免疫3次，首免时将p54蛋白与弗氏完全佐剂混匀乳化（150 μg/只），二免、三免使用弗氏不完全佐剂。三免7 d后对小鼠进行尾部采血，使用ELISA方法测定小鼠血清抗体效价，选取效价高的小鼠进行加强免疫。3 d后取小鼠脾细胞与SP2/0细胞进行融合，利用重组p54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mAb细胞株，并制备腹水。以pCAGGS-Flag-E183L质粒转染的HEK293T细胞和ASFV Pig/HLJ/18株感染的猪肺泡巨噬细胞（PAMs）为抗原，以筛选得到的mAb为一抗，进行Western blot鉴定和间接免疫荧光试验（IFA）。使用单抗亚类鉴定试剂盒检测该mAb重链和轻链类型。随后，将p54蛋白逐步截短并与GST标签融合表达后进行Western blot检测，鉴定该mAb识别的抗原表位。【结果】将构建的原核表达质粒pET-21a-E183L（54-183aa）转化至BL21（DE3）感受态细胞中，使用IPTG进行诱导后，p54重组蛋白大部分以可溶的形式在上清中表达，分子量约为17 kDa。以纯化得到p54重组蛋白免疫小鼠，三免后7 d的血清效价为1﹕409 600，可进行细胞融合，经四次筛选和亚克隆后，获得能够稳定分泌p54蛋白mAb的细胞株，命名为5B11，成功制备腹水并纯化。Western blot和IFA试验结果显示，制备的mAb能够特异性识别HEK293T细胞中表达及ASFV感染PAMs中的p54蛋白。mAb亚类鉴定结果显示重链类型为IgG1型，轻链类型为κ链。该mAb识别的抗原表位序列为⁸⁰VTPQPGTSKPA⁹⁰。【结论】利用原核表达体系可溶性表达了ASFV p54蛋白的第54-183位氨基酸,并以纯化的蛋白为抗原制备了抗p54蛋白的mAb，并对其识别的抗原表位进行鉴定，丰富了p54蛋白的抗原表位信息，为ASFV血清学检测提供了基本材料。

关键词: 非洲猪瘟病毒, p54蛋白, 单克隆抗体, 抗原表位

Abstract:

【Objective】The aim of this study was to obtain soluble African swine fever virus (ASFV) p54 protein and anti-p54 monoclonal antibodies (mAb), along with the identification of their recognized epitopes, so as to lay a foundation for the study of the structure and function of p54 protein and the development of serological diagnostic reagents. 【Method】To prepare mAb against ASFV p54 protein, the prokaryotic recombinant expression plasmid pET-21a-E183L was constructed and transformed into E.coli BL21 (DE3) cells. The recombinant protein p54 was purified by Ni-affinity chromatography and gel filtration, five weeks old BALB/c mice were immunized with the purified p54 recombinant protein. The immunization protocol involved three rounds at two-week intervals, starting with an emulsion of the antigen and an equal volume of Freund’s complete adjuvant, followed by the same antigen emulsion with an equal volume of Freund’s incomplete adjuvant for the subsequent immunization. After three immunizations, the blood of mice was collected, and the serum antibody titers were detected by indirect enzyme-linked immunosorbent assay (ELISA). The mice with the highest serum titers were selected for a booster immunization. Spleen cells from the mice were fused with SP2/0 cells three days later. The positive hybridoma cells were screened by indirect ELISA with the recombinant p54 protein. The specificity of the selected mAb was further confirmed via Western blot and indirect immunofluorescence assay (IFA). The isotype of the mAb was detected using a subclass identification kit. Subsequently, the p54 protein was systematically truncated and expressed as GST fusion proteins to identify the antigen epitopes recognized by the mAbs by Western blot. 【Result】 The constructed prokaryotic expression plasmid pET-21a-E183L (54-183 aa) was transformed into BL21 (DE3) cells. After induction with IPTG, the p54 recombinant protein was expressed in the supernatant in a soluble form with a molecular weight of about 17 kDa. Immunization of mice with purified p54 recombinant protein results in a serum titer of 1﹕409 600 seven days after the third immunization. Cell fusion was successfully performed. After four rounds of subcloning, a hybridoma cell line named 5B11 that could stably secrete mAb against p54 protein was obtained. The ascites fluid was prepared and the mAbs were purified. Western blot and IFA assay results showed that 5B11 could specifically recognize p54 protein expressed in HEK293T cells and ASFV-infected porcine alveolar macrophages (PAMs). The mAb subclass identification showed that 5B11 was of IgG1 heavy chain and κ light chain. The epitope sequence recognized by 5B11 was ⁸⁰VTPQPGTSKPA⁹⁰.【Conclusion】In this study, the recombinant protein ASFV p54 with amino acid 54-183 was successfully expressed in a soluble form in the prokaryotic system. The development of anti-p54 mAbs and the identification of their recognized epitopes have expanded understanding of p54 protein epitopes and provided the basic materials for the serological detection of ASFV.

Key words: African swine fever virus, p54 protein, monoclonal antibody, epitope

冯春莹, 张朝霞, 刘云飞, 黄丽, 翁长江. 非洲猪瘟病毒p54蛋白单克隆抗体的制备及其抗原表位鉴定[J]. 中国农业科学, 2024, 57(19): 3936-3944.

FENG ChunYing, ZHANG ZhaoXia, LIU YunFei, HUANG Li, WENG ChangJiang. Preparation of Monoclonal Antibody Against African Swine Fever Virus p54 Protein and Identification of Its Epitope[J]. Scientia Agricultura Sinica, 2024, 57(19): 3936-3944.

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