Abstract
BackgroundPorcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which therefore is an ideal target for the development of subunit vaccine. In an attempt to develop a subunit vaccine for PEDV, we cloned two different fragments of S protein and expressed as glutathione S-transferase (GST)-tagged fusion proteins, namely rGST-COE and rGST-S1D, in E.coli. However, the expression of these recombinant protein antigens using a variety of expression vectors, strains, and induction conditions invariably resulted in inclusion bodies. To achieve the soluble expression of recombinant proteins, several chaperone co-expression systems were tested in this study.ResultsWe firstly tested various chaperone co-expression systems and found that co-expression of trigger factor (TF) with recombinant proteins at 15 °C was most useful in soluble production of rGST-COE and rGST-S1D compared to GroEL-ES and DnaK-DnaJ-GrpE/GroEL-ES systems. The soluble rGST-COE and rGST-S1D were purified using glutathione Sepharose 4B with a yield of 7.5 mg/l and 5 mg/l, respectively. Purified proteins were detected by western blot using mouse anti-GST mAb and pig anti-PEDV immune sera. In an indirect ELISA, purified proteins showed immune reactivity with pig anti-PEDV immune sera. Finally, immunization of mice with 10 μg of purified proteins elicited highly potent serum IgG and serum neutralizing antibody titers.ConclusionsIn this study, soluble production of recombinant spike protein of PEDV, rGST-COE and rGST-S1D, were achieved by using TF chaperone co-expression system. Our results suggest that soluble rGST-COE and rGST-S1D produced by co-expressing chaperones may have the potential to be used as subunit vaccine antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-016-0268-7) contains supplementary material, which is available to authorized users.
Highlights
Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine
Chaperone assisted expression of soluble rGST-COE and rGST-S1D Based on the sequence information of the spike protein of PEDV CV777 strain, we constructed pG-COE and pG-S1D expression plasmids, producing rGST-COE [17] and rGST-S1D [13], respectively
Unlike the previous reports [13, 17], our initial attempts to produce rGSTCOE and rGST-COE using a pGEX 6p-1 vector system with various induction conditions resulted in inclusion bodies (IBs)
Summary
Porcine epidemic diarrhea virus (PEDV) is a highly contagious enteric pathogen of swine. The spike glycoprotein (S) of PEDV is the major immunogenic determinant that plays a pivotal role in the induction of neutralizing antibodies against PEDV, which is an ideal target for the development of subunit vaccine. Porcine epidemic diarrhea (PED) is a highly infectious and contagious enteric disease of swine [1]. A coronavirus named porcine epidemic diarrhea virus (PEDV) was identified as the causative agent of PED in the late 1970s [2]. A novel neutralizing epitope region S1D (aa 636–789) on the S1 domain was reported to have the capacity to induce neutralizing antibodies against PEDV [13]. Spike protein is considered as a primary target for the development of subunit vaccine against PEDV
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