4. Discussion

Since ASF was first reported in Kenya in 1921, it caused high mortality in domestic pigs(Davies et al., 2017). African swine fever (ASF) is the most serious epidemic disease in the pig industry, which is caused by the African swine fever virus (ASFV). Monoclonal antibodies (mAb) are key reagents for diagnostic detection of viral infection(Neilan et al., 2004). ASFV p30 protein is an early phosphorylated protein encoded byCP204L gene which is one of the most immunogenic proteins(Agüero et al., 2003; Zhou et al., 2018).
Monoclonal antibodies against ASFV p30 protein are powerful tools for mapping ASFV p30 protein epitopes and investigating antigenic(Petrovan et al., 2019). Undoubtedly, the gold standard for epitope definition is to determine the 3D structure of the antigen-antibody complex by X-ray or cryo-EM, but it is not readily applicable to many antigens and antibodies, for its laborious efforts with a low success rate(Gershoni, Roitburd-Berman, Siman-Tov, Tarnovitski Freund, & Weiss, 2007). A second approach to epitope mapping uses nuclear magnetic resonance (NMR) which gives a picture of the antigen-antibody complex in solution. Other means of epitope mapping include computational docking, binding analyses, alanine scanning mutagenesis (ASM), and saturating mutagenesis(Huynen, Filée, Matagne, Galleni, & Dumoulin, 2013). B-cell epitopes are classified as linear or conformational, though it has been reported that 90% of B cell-recognizing epitopes are conformational epitopes, the antigen internalizing process and antigen recognizing ability(Barlow, Edwards, & Thornton, 1986; Chang et al., 2019; Van Regenmortel MHV, 1996). Different methods have been used for the identify-cation of epitopes.
Escherichia coli (E. coli) is a remarkable host due to its rapid growth rate, requirement for inexpensive carbon sources, low cost and well-characterized genetic structure(Malakar & Venkatesh, 2012; Overton, 2014; Scaglia, Cassani, Pilu, & Adani, 2014). However, E. coli has some defects such as inability for posttranslational modifications, ineffective cleavage of the amino terminal methionine, inability to produce proteins containing complex disulfide bonds, and expression of proteins as insoluble inclusion bodies (IBs) (IBs). Thus, in our study , we choose three molecular chaperone to improve the the soluble expression of the target protein. A fundamental function of molecular chaperones is to inhibit unproductive protein interactions by recognizing and protecting hydrophobic surfaces that are exposed during folding or following proteotoxic stress(Balchin, Hayer-Hartl, & Hartl, 2020; Mamipour, Yousefi, & Hasanzadeh, 2017). we constructed three co-expression system with chaperone plasmids. After purified by His Trap FF and His Trap Q HP affinity column, obtain the pure protein. Monoclonal antibodies were produced against purified p30 protein and their epitopes investigated. The three antibodies we tested, all recognized the N-terminal amino acids of p30 protein, indicating that this section contains the main antigenic epitope of p30 protein.
In the present study, Vlad Petrovan, Fangfeng Yuan used the prokaryotically expressed p30 protein as immunogen to get three mAbs, and two of the mAbs recognize the C-terminal region of the protein, the other one could recognized a linear B cell epitope 61-93 amino acid(Petrovan et al., 2019). Wu, Lowe, Rodriguez et al developed a panel of 21 mAbs against ASFV p30. With 14 out of the 21 mAbs, they defined 4 antigenic regions that contain at least 4 linear epitopes. Four linear epitopes contain 61-90aa, 96-115aa, 111-130aa, 143-160aa(Wu et al., 2020). Zhang, Liu, Wu, et al generated two mAbs against the ASFV p30 and found two novel linear B cell epitopes, 144-154 aa and 12-18 aa(Zhang et al., 2021).