Introduction
Hand-foot-mouth disease (HFMD) is an infectious disease caused by enterovirus 71 (EV71). The virus is an important pathogenic factor of hand, foot and mouth disease. Vp1 protein is the viral capsid protein and promotes the infection of host cells by virus particles. Vp1 is also the main antigen gene of the EV71 virus. Generally, the vaccinated population, especially infants and young children, are more compliant with oral vaccines, so we are trying to develop oral HFMD vaccines. Probiotics bacteria Bifidobacteria, as the natural host of the intestinal tract, can adhere to intestinal epithelial cells and are ideal oral live vaccine expression vectors, and related studies have found that their preventive effects on gastrointestinal pathogens are more significant. Therefore, we can use the bifidobacterium in lactic acid bacteria as an expression system to express EV71 vp1.
Design
The B subunit LTB in the heat-labile enterotoxin (LT) of Escherichia coli heat-labile enterotoxin (LT) has strong immunogenicity and adjuvant activity, and will not cause harm to the human body. LTB and a variety of non-related proteins and their non-protein antigens can increase the mucosal IgA and humoral immune IgG response levels of the antigen through different immunization pathways. Fusion recombinant protein constituting EV71 vp1 and LTB is produced by bifidobacterium expression system. Vp1-LTB is tagged by glutathione S-transferase (GST) in its N-terminal. The tag is used for recombinant protein purification.
Build
Plasmids pGEX-vp1 and pGEX-vp1-LTB were constructed to produce Vp1 and Vp1-LTB fusion proteins in probiotics bacteria Bifidobacterium, respectively (Figure 1). The production is controlled by tac promotor. The construct is confirmed by DNA sequencing.
Figure 1. Schematic map of plasmids pGEX-vp and pGEX-vp1-LTB.
Test
Due to the relatively low rate of growth and efficiency of electroporation of L. casei, our team first transformed E. coli BL21, which is commonly used in plasmids transformation, to verify the expression and antigencity of VP1 and VP1-LTB proteins (Figure 2).
Figure 2. SDS-PAGE and Western Blot for expression of VP1 and VP1-LTB proteins.
Learn
GST, GST-VP1 and GST-VP1-LTB had all been successfully expressed by E.coli BL21, and they mainly existed in the precipitation in the form of inclusion body. The expression of VP1 and VP1-LTB was not so effective in E.coli BL21. There was no obvious expression of GST-VP1 and GST-VP1-LTB in L. casei.