A novel multi-epitope recombinant protein for diagnosis of African swine fever

Background: African swine fever(ASF) is an acute, severe and highly fatal infectious disease of pigs. The disease spreads rapidly, causing huge economic losses to the pig industry in infected areas. The structural proteins p30 and p54 in African swine fever virus(ASFV) have been verified as diagnostic antigens. Methods: In this study, we constructed a novel multi-epitope fusion antigen gene based on P30 and P54 proteins, induced expression in a prokaryotic expression system and analyzed the reactivity of the recombinant fusion protein. The purified recombinant protein m35 was used as the coating antigen to establish an indirect enzyme-linked immunosorbent assay (ELISA) detection method for ASFV. 116 serum samples and positive sera of other swine diseases were detected by indirect ELISA. Results: Our results indicate that the m35 gene fragment with a length of 558bp was successfully constructed. SDS-PAGE and Western Blotting analysis showed that the protein had a band at 22kDa, proving its good reactogenicity. ROC analysis was performed to validate the assay, the area under the ROC curve is 0.9738 (95% confidence interval, 0.9336 to 1.014), and does not cross-react with other swine diseases. Conclusion: Our results show that its sensitivity and specificity were highly accurate. It is feasible to use this recombinant protein as a diagnostic antigen to distinguish ASFV infection.

variants between other gene type II strain This strain has the highest similarity to the POL / 2015 / Podlaskie strain(99.972%) [5]. No commercial vaccines are currently available, so establishing effective detection methods is of great significance for the prevention and control of ASF.
ASFV is composed of five parts: nucleoid, core shell, inner envelope, capsid, and external envelope. It encodes 54 structural proteins and more than 100 non-structural proteins [4]. Both P30 and P54 proteins in structural proteins can induce specific immune responses. The P30 protein encoded by the open reading frame CP204L is mainly involved in virus internalization and plays an important role in the process of the virus entering the host cell [13]. And a large number appeared in the early stage of virus infection, inducing the body to produce neutralizing antibodies [1]. The P54 protein encoded by the E183L gene is present in the inner envelope of the virions and is involved in the adsorption and entry of the virus. Therefore, P30 and P54 proteins are ideal antigens for serological diagnosis and immunological detection [11,15].
In this study, we predicted the predominant epitopes of P30 and P54 proteins to synthesize the synthetic gene m35 with small molecular weight and easy expression. The prokaryotic expression of the gene was purified, and the immunological characteristics of the recombinant protein were analyzed by SDS-PAGE and western blotting, and an indirect ELISA detection method based on the recombinant protein was established; this provided an effective way for the timely diagnosis of ASFV.

Expression, and purification of recombinant protein
The synthetic recombinant plasmid was transformed into E. coli BL21(Sangon Biotech Co., Ltd Shanghai China). The transformed E. coli BL21 cells were grown in a shaker at 220 rpm and 37 °C, inoculated into 4 L of LuriaBertani (LB, containing 30 μg/ml kanamycin) at a dilution of 1:100. When the optical density at 600 nm (OD600) of the culture reached approximately 0.6, the culture was induced by 1M IPTG. Induction at 37°C for four hours, the cell culture was collected by centrifugation, and aliquots before and after induction were analyzed by SDS-PAGE. The collected cell culture was resuspended in buffered lysate and then sonicated on ice. The lysate was centrifuged at 5000g and 4℃ for 20 minutes, and binding buffer containing 8 M urea was added to the precipitate (inclusion body form) for overnight dissolution at 4℃. After centrifugation, the recombinant proteins containing the His-tag were purified by affinity chromatography using Ni SepharoseTM excel (GE). The purified proteins were refolded in dialysate (0.25 M NaCl, 50 mM Tris, 0.5 mM EDTA, 2 mM GSH, and 0.2 mM GSSG; pH 8.0) containing different concentrations of urea (6 M to 0 M in 1 M decrements) at 4℃. Finally, the proteins were dialyzed in PBS (pH 7.4) twice to obtain the completely renatured proteins. The concentration of m35 protein was determined using the Bradford Protein Assay Kit, respectively, according to the manufacturer's instructions.

SDS-PAGE, Western blot analysis
The purified proteins m35 were analyzed by 10% SDS-PAGE and stained with Coomassie blue R250 or transferred to a polyvinylidene fluoride (PVDF) membranes for Western blot analysis using a mouse anti-His monoclonal antibody (mAb; Abcam, MA) and the ASFV positive serum. The membranes were blocked in PBST containing 5% skim milk at 37℃ for 1 hour, and then incubated overnight at 4℃ with diluted mAb or positive serum. The membranes were then washed three times with PBST and incubated with the diluted goat antimouse and rabbit anti-pig second antibody conjugated with horseradish peroxidase (HRP; Abcam) at 37℃ for 1 hour. After three washes in PBST, protein detection was visualized with the BeyoECL Plus Kit (Beyotime, China), following the manufacturer's instructions, and exposed to X-ray film. Each band was analyzed using a Gel Image System(Tanon, China). iELISA Ninety-six-well microtiter plates (Corning, USA) were coated with 0.25 μg per well of the recombinant protein in coating buffer (0.01 M PBS, pH 7.4) and incubated overnight at 4 °C. Incubate overnight. After washing 3 times with PBST, blocking with 5% skimmed milk powder at 37 ° C for 2 h, and then washing 3 times, incubated with serum 1:100 in serum dilution(100μL per well) at 37 ° C for 1 hour, then wash the wells 3 times, and dilute anti-swine IgG antibody to 1: 20000, add 100 μL per well, incubate for 30 minutes at 37 ° C. After washing 3 times with PBST, 100 μL of TMB substrate was added to each well. After reacting at 37 ° C for 15 minutes, 100 μL per well of stop solution (2M H2SO4) was added. ODs were measured at 450 nm using an ELISA plate reader (BioTek,USA).
Dot plot and receiver-operating characteristic (ROC) were performed using the GraphPad Prism version 7.0 for Windows. 116 serum samples from Professional laboratory in Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu, China. Serum samples were classified as negative or positive according to Western blot results, with 87 negative samples and 29 positive samples.

Cross-reaction analysis
The examination of the analytical specificity by m35 ELISA was conducted for determining the cross-reactive degree among PRV, PRRSV, PCV, PDCoV, CSFV, and FMDV sera samples. The experiment contained negative control and positive control.

Construction of multiple epitope genes
The Chinese strain 2018 / AnhuiXCGQ genome's genebank is MK128995.1. Then integrate multiple epitope prediction programs to select the cell epitope with the best overlap result, as shown in Table1. Recombinant protein identification we inserted the synthetic gene in the bacterial expression vector pET-28a and transformed it into E. coli BL21 (DE3) cells. Then, the induced cells were sonicated and analyzed by SDS-PAGE. A specific band representing the recombinant product was obtained ( Fig. 1A and B). Ni-NTA affinity purification was used, and the purified products were analyzed by SDS-PAGE (Fig. 1c). The concentration of the product was 1.4 mg/ml when measured with a protein assay kit. Western blot analysis showed that m35 could react with anti-His mAb (Fig. 2W1), as well as ASFV positive serum (Fig.2W2). To evaluate the analysis, 116 serum samples were tested by indirect ELISA and Western blot, including positive serum (29 samples) and negative serum (87 samples). The dot plot summarizes the OD values of these samples (Fig. 3a).ROC analysis was performed to assess the best sensitivity and specificity (Fig. 3b). According to ROC analysis, the AUC for this test was 0.9738 (95% confidence interval (CI) 0.9336 to 1.014). In addition, a diagnostic sensitivity of 93.1% (95% CI, 77.23 to 99.15) and a specificity of 96.55% (95% CI, 90.25 to 99.28) were obtained from the optimal cutoff value (0.3435).

Cross-reaction analysis
There were no cross reactions in the ELISA using PRV, PRRSV, PCV, PDCoV, CSFV, and FMDV positive sera. The OD values of the disease-positive sera were less than their critical values (Fig. 4).

Discussion
The current laboratory diagnosis of African swine fever includes animal inoculation, virus isolation, viral nucleic acid DNA detection, and specific antibody detection. Among them, animal vaccination, virus isolation, and viral nucleic acid detection require professional personnel in laboratories above Level 3 The operation is proceeding, it is difficult to meet the needs of the grassroots. Therefore, the serological detection method is one of the commonly used diagnostic methods for ASFV due to the simple operation steps and low cost. It is one of the important methods for diagnosing and monitoring pigs infected with African swine fever virus [14].
In serological diagnosis, In 1979, ELISA was used for the first time to identify ASFV antigens and antibodies. The whole virus antigens were used. The reproducibility was good and a limited antigen concentration of 50-500 HAD50 / ml could be detected [19]. p72, p30, p54, pp62 are the major diagnostic proteins [6,9]. P72 protein is the main capsid protein, mainly involved in virus binding cells [13]. Since the semi-purified P72 protein was prepared in 1981 to establish an ELISA method, the protein has been used for detection research [17]. There are currently commercial detection kits based on this protein. PP62 protein is mostly identified as a late-stage protein. After proteolytic processing, two main structural proteins, p35 and p15, are produced [16]. The expression of pp62 using the baculovirus expression system and the establishment of an ELISA detection method show that the sensitivity and specificity are improved compared to traditional diagnostic methods [8]. P30 and p54 proteins are the most antigenic proteins [2]. Both proteins are incorporated into virus particles and mediate vaccine neutralization [12]. Interestingly, p54 protein has better reactivity than p30 in protein imprinting, but recombinant p30 protein is more effective in detecting antibodies by ELISA. This may be related to the conformational epitope included in p30 [15]. Since then, these two proteins have been widely used in the diagnosis of African swine fever [3,15].
The B cell epitope refers to the chemical group on the surface of the antigen molecule that can be specifically recognized and bound by the b cell surface receptor or antibody Fab part, which is the material basis for humoral immunity. Is the focus of research in immunodiagnosis and immunotherapy [18]. In recent years, with the development of bioinformatics, the bioinformatics prediction of epitopes has gradually replaced the traditional methods of epitope research. At present, the prediction of B cell linear epitopes is mainly based on the physical and chemical properties, structural characteristics, and statistical significance of amino acids. In our research, we selected the highly immunoreactive p30 and P54 proteins. We used DNAStar, ABCpred, Scratch, and IEDB to perform linear b-cell epitope analysis and prediction of these two protein sequences. In the end, we chose 10 epitopes as a result. The GGGGS flexible linker is used to connect each epitope in series, and the research proves that the flexible linker is more conducive to the function of each epitope. This is used as a new antigen sequence for detecting ASFV for prokaryotic expression and purification. The Western blotting analysis results show that m35 protein has good antigenicity and is suitable for diagnostic antigens ROC analysis was performed to validate the assay, and its sensitivity and specificity were highly accurate(0.9<AUC<1). And after testing, m35 iELISA does not react with other swine diseases. Therefore, it is feasible to use m35 recombinant protein as a diagnostic antigen to distinguish ASFV infection.
However, although the Elisa method is simple and suitable for large-scale epidemiological surveys, it is not suitable for the diagnosis of ASFV during the outbreak period, many influencing factors will appear false positive.

Conclusions
In this article, we analyzed and predicted the structural proteins p30 and p54 of the African swine fever virus with software analysis, selected the dominant epitope fragments, and connected them with flexible peptides to obtain the recombinant gene m35. And confirmed that the indirect ELISA method based on m35 protein has high sensitivity and specificity, which provides a choice for the development of the ASFV Elisa serological detection kit.