Recombinant Human SCARB2 Expressed in Escherichia coli and its Potential in Enterovirus 71 Blockage

Iran J Sci Technol Trans Sci https://doi.org/10.1007/s40995-020-01025-9 (0123456789().,-volV)(0123456789(). ,- volV) RESEARCH PAPER Recombinant Human SCARB2 Expressed in Escherichia coli and its Potential in Enterovirus 71 Blockage Hai-Vy Vo-Nguyen1,2 • Thanh-Tan Nguyen1,2 • Huyen-Trang Thi Vu3 • Thanh-Thao Thi Nguyen3 • Quoc-Cuong Hoang3 • Thuoc Linh Tran1,2 • Hieu Tran-Van1,2 Received: 27 April 2020 / Accepted: 2 November 2020 Ó Shiraz University 2021 Abstract Hand, foot and mouth disease is a common viral infectious disease caused by enteroviruses, including coxsackie A16 (CVA16) and enterovirus 71 (EV71). HFMD can cause severe symptoms in children which can be fatal. Human scavenger receptor class B member 2 (SCARB2) is a cellular receptor for EV71 and CVA16, providing a potential approach for preventing EV71 infection and transmission. In this present study, we constructed and assessed the potential of recombinant SCARB2, using E. coli expression system. To generate this construct, scarb2 gene was cloned into pET22b vector and expressed in E. coli BL21 (DE3). The expression of SCARB2 was induced by 0.1 mM IPTG and analyzed using SDSPAGE, followed by Western blot. Expressed SCARB2 was in inclusion bodies and refolded to obtain the soluble form with recovery efficacy of 100%. This recombinant protein was then validated for binding with EV71 via indirect ELISA in two different pHs (7.4 and 5.5), which partially revealed the mechanism of virus–receptor interaction. These results envisaged potential applications for utilizing recombinant SCARB2 in preventing the virus transmission. Keywords EV71  HFMD  Inclusion bodies  Refolding  SCARB2 1 Introduction Hand, foot and mouth disease (HFMD) is a common viral infectious disease characterized by common symptoms including fever, painful sores in the mouth, and a rash with blisters on hands, feet and buttocks. In most cases, the disease is mild and self-limiting, occurring mainly in children under 10 years old, but it is most commonly seen in children under 5 years of age. It can also appear in adolescents and occasionally in adults. Viruses causing HFMD are spread by direct contact with saliva, mucus, fluid from blisters and stool of infected people. Adults infected with HFMD may shed the virus and have no symptoms (WHO). The dominant cause of HFMD is a & Hieu Tran-Van 1 Faculty of Biology and Biotechnology, University of Science, Ho Chi Minh City, Vietnam 2 Vietnam National University, Ho Chi Minh City, Vietnam 3 Pasteur Institute, Ho Chi Minh City, Vietnam group of enteroviruses, including coxsackie A16 (CVA16) and enterovirus 71 (EV71) (Yamayoshi et al. 2012a). Although complications are uncommon, EV71 infection can lead to severe symptoms in children and has been associated with neurological diseases, including acute flaccid paralysis, aseptic meningitis, and brain stem encephalitis with neurogenic pulmonary edema, which can be fatal (Lee 2016). There are no specific antiviral drugs or vaccine commercially available against enteroviruses causing HFMD hitherto (WHO). EV71 belongs to the human enterovirus A species of the enterovirus genus within the family Picornaviridae (King et al. 2000). Virion includes a non-enveloped capsid surrounding a core of single-stranded, positive-polarity RNA approximately 7.5 kb in size (Brown and Pallansch 1995). Since its discovery in 1969, EV71 has been recognized as a frequent cause of epidemics of HFMD associated with severe neurological sequelae in a small proportion of cases (WHO 2011). Approaches have been intensively evaluated to prevent the spread of the virus, in which vaccines are the most priority (Yi et al. 2017). Several significant milestones 123 Iran J Sci Technol Trans Sci have been archived recently in vaccine development of China, which leads to its phase 3 testing on trial (Li et al. 2014; Zhu et al. 2013, 2014). In December 2015 and January 2016, two HFMD vaccines approved by the China Food and Drug Administration were commercially produced and began to apply on Chinese children. However, these vaccines required long duration to demonstrate their efficacy and safety before becoming commercially available for application (Yi et al. 2017). In addition, the outreach efforts toward preventing the virus from entering its target cells also achieved certain results. Many studies were conducted to identify receptors on the surface of human cells that play a role in facilitating the infection of EV71. In particular, SCARB2 receptor presents in many cell types in the body and has been shown to be an important factor in the infection of EV71, which can be bound by all existing EV71 strains (Yamayoshi et al. 2009). Moreover, SCARB2 is also a co-receptor for CVA16, one of the main causative agents of HFMD besides EV71 (Yamayoshi et al. 2012b). SCARB2 has been confirmed to have ten potential N-glycosyl binding sites, considered to be responsible for its proper folding (Yamayoshi and Koike 2011). Recently, recombinant receptor produced in E. coli has been shown to interact with EV71 in vitro (Xu et al. 2018). However, the receptor was used in insoluble form, so it is not suitable for related or further research, as well as putting into applications. On the other hand, the role of the receptor’s glycosylation remained unclear in interaction with EV71. In this study, we created a SCARB2-expressing plasmid to produce recombinant receptor in E. coli along with a refolding procedure. Then, the interaction between recombinant SCARB2 and EV71 was evaluated by indirect ELISA. utilized for band size marking. Recombinant protein was constructed, expressed and stored at -200C. 2.2 Construction of Recombinant pET-SCARB2 (Fig. 1) pPHAGE-C-TAP-SCARB2 plasmid was used as a template for amplification of scarb2 gene by PCR with specific primers (scarb2F: CATATGatcgagaagaaaattgtg and scarb2R: CTCGAGaatcatagacttcagtcgac). scarb2 gene and pET22b plasmid were digested with NdeI and XhoI (Thermo Scientific). The ligation reaction containing the double-digested insert and expression vector was performed in the presence of T4 DNA ligase (Thermo Scientific), and the resulting mixture was transformed into competent E. coli DH5a. The transformants were initially screened on ampicillin-containing LB agar plate and then re-screened by PCR with specific primers and T7pro/T7ter. Plasmids derived from positive colonies confirmed by PCR colonies were sent to the Macrogen, Korea, for sequencing. 2.3 Expression of Recombinant SCARB2 in E. coli BL21 (DE3) The expression of recombinant SCARB2 was conducted as described with some modifications (Seidmoradei et al. 2020). pET-SCARB2 obtained from previous steps was transformed into competent E. coli BL21 (DE3) strain. Positive colonies were inoculated in LB media shaking tubes supplemented with ampicillin and allowed to grow at 37 °C in 16 h. The cultures were then sub-cultured at 1:10 (v/v) and inoculated at 37 °C until OD600 reached 0.8–1.0. 2 Ma (...truncated)