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).