Identification of a conservative site in the African swine fever virus p54 protein and its preliminary application in a serological assay

Abstract

Background: ASF was first reported in Kenya in 1910 in 1921. In China, ASF spread to 31 provinces including Henan and Jiangsu within six months after it was first reported on August 3, 2018. The epidemic almost affected the whole China, causing direct economic losses of tens of billions of yuan. Cause great loss to our pig industry. As ELISA is cheap and easy to operate, OIE regards it as the preferred serological method for ASF detection. P54 protein has good antigenicity and is an ideal antigen for detection.

Objective: To identify a conservative site in the African swine fever virus (ASFV) p54 protein and perform a Cloth-enzyme-linked immunosorbent assay (ELISA) for detecting the ASFV antibody in order to reduce risks posed by using the live virus in diagnostic assays.

Method: We used bioinformatics methods to predict the antigen epitope of the ASFV p54 protein in combination with the antigenic index and artificially synthesized the predicted antigen epitope peptides. Using ASFV-positive serum and specific monoclonal antibodies (mAbs), we performed indirect ELISA and blocking ELISA to verify the immunological properties of the predicted epitope polypeptide.

Results: The results of our prediction revealed that the possible antigen epitope regions were A23-29, A36-45, A72-94, A114-120, A124-130, and A137-150. The indirect ELISA showed that the peptides A23-29, A36-45, A72-94, A114-120, and A137-150 have good antigenicity. Moreover, the A36-45 polypeptide can react specifically with the mAb secreted by hybridoma cells, and its binding site contains a minimum number of essential amino acids in the sequence 37DIQFINPY44.

Conclusions: Our study confirmed a conservative antigenic site in the ASFV p54 protein and its amino acid sequence. A competitive ELISA method for detecting ASFV antibodies was established based on recombinant p54 and matching mAb. Moreover, testing the protein sequence alignment verified that the method can theoretically detect antibodies produced by pigs affected by nearly all ASFVs worldwide.

Keywords: African swine fever virus; antigenic site; conservative; monoclonal antibody.

Fig. 1. Results of SDS-PAGE and western blot analysis for detecting the prokaryotic expression of the ASFV p54 protein. (A) SDS-PAGE analysis (M: protein marker; 1: positive control; 2: negative control). (B) Western blot positive hybridization.

Fig. 2. Results of a western blot analysis to detect the eukaryotic expression of the ASFV p54 recombinant protein (M: protein marker; 1: normal Sf9 cell protein; 2: p54 recombinant protein).

Fig. 3. Analysis of the characteristics of the p54 protein’s secondary structure.

Fig. 4. Results of a blocking ELISA for polypeptide binding reactions with mAb.

ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody.