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.