Introduction
Bluetongue (BT) is a non-contagious, infectious, World Organisation for
Animal Health (OIE) notifiable disease of ruminants caused by the
Bluetongue virus (BTV, family Reoviridae, genus Orbivirus) and is spread
by Culicoides spp. biting midges (Coetzee, Stokstad, Venter, Myrmel, &
Van Vuuren, 2012; Kar, Ghosh, & Roy, 2004; Mokoena et al., 2019; Schulz
et al., 2016). There are 27 distinct known BTV serotypes determined by
the outer viral-capsid protein VP2 encoded by segment 2 of the dsRNA
genome (Coetzee et al., 2012; Mokoena et al., 2019; Schulz et al.,
2016). Furthermore, BTV infected sheep often show severe clinical signs,
while cattle, goats and camelids are usually asymptomatic, although some
clinical cases in cattle have been observed during the North European
outbreak of BTV-8 (Backx, Heutink, van Rooij, & van Rijn, 2007;
Caporale et al., 2014; Dal Pozzo, De Clercq, et al., 2009; Dal Pozzo,
Saegerman, & Thiry, 2009; Eschbaumer et al., 2011). The BTV particle is
a non-enveloped, complex virus of two capsids. The outer capsid is
composed of VP2 and VP5. The inner capsid (named as “core”) has two
concentric layers in which the first layer is formed by VP3,and the
other by VP7. Both VP3 and VP7 are highly conserved across all BTV
serotypes (Kar et al., 2004; Roy, 2008; Stewart et al., 2012).
Commonly used BTV protein expression methods mainly include eukaryotic
expression and prokaryotic expression. Xie (Xie et al., 2018) expressed
the structural protein VP2 of BTV-25 in sf9 insect cells with
baculovirus, and Hassan (Hassan, Wirblich, Forzan, & Roy, 2001) used
the baculovirus to express the structural protein VP5 in sf9 insect
cells. Many studies express VP3 by inclusion bodies or truncated
methods. Wang (Wang et al., 2013) expressed a truncated version of VP5
which lacked the first 41 a.a., because the expression level of
full-length recombinant VP5 protein in E.coli BL21 was very low.
The easiest, quickest, and cheapest technique in expression of proteins
is the use of E.coli , that has been widely employed in industrial
biotechnology for a long time (Hayat, Farahani, Golichenari, &
Sahebkar, 2018). The high safety compared with that in other organisms,
as well as need for cheap media and simple growth conditions (37°C)
(Rosano & Ceccarelli, 2014).
Although great progress has been made in heterologous protein expression
in E. coli , the expression of proteins with optimal solubility
and appropriate structural and functional properties remains a problem
(Hayat et al., 2018). Among various approaches to alleviate protein
aggregation, it is widely recognized that the coexpression of molecular
chaperones and fusion tag technology can assist with protein folding,
which leads to an increased production of active protein (Lee, Kim,
Jeong, & Lee, 2002; Stewart et al., 2012). In this study, the molecular
chaperone pTf16 and two fusion proteins, TRX and NusA, were selected to
co-express with the VP3 in order to increase the supernatant expression
of the VP3 (Figure 1). The purpose of the study was to improve the
solubility of the VP3, and then analyze the immunogenicity. To lay the
foundation for further research on the structure and function of BTV
protein VP3.