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

Abstract

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

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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References 32

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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

Preparation of Monoclonal Antibodies Against the p30 Protein of African Swine Fever Virus and Its Mapping of Linear Epitopes

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