Biological Characteristics Of The Complete And Truncated VP1 Protein Of Enterovirus 71

Enterovirus 71 (EV71) is a member of the enterovirus genus of the Picornaviridae family. Children under 5 years old are constantly involved with hand-foot-mouth disease (HFMD) with lesions in central nervous system and pulmonary edema. There are still no specific anti-EV71 drugs in the market while the vaccine has already got initially positive results. On December 3rd,2015, the first EV71 inactivated vaccine in the world was approved by China Food and Drug Administration. This kind of vaccine could protect 80% children from the EV71 infection with decent safety and efficacy according to the clinical trials. However the potential drawbacks of inactivated vaccines such as low immunity, short duration of the immune response and the multiple inoculations to complete the immunization process remind us that the developement of efficacious vaccine is in urgent need in the prevention for the EV71 infection. Peptide vaccine is a new approach for vaccine that is mixtures of epitopes that induce protection against infection and appropriate adjuvants and vectors. The utilization of peptide vaccine could realize the comprehensive guard against the virus and avoid the recovery of the virulence compared with attenuated and inactivated vaccines. The genome of EV71 encodes 4 structural protein (VP1, VP2, VP3 and VP4) and the main epitope loacates in the VP1. In this study, structural protein VP1 and the truncated VP1 proteins of EV71 were successfully expressed in prokaryotic expression system. The biological activities of VP1 protein and polypeptides of truncated VP1 were then analyzed. Our research laid the foundation for the development of the subunit vaccines of peptide fragments.Objective1. To construct the prokaryotic expression vector of EV71 structural protein VP1 and truncated polypeptides of VP1.2. To express and purify the EV71 structural protein VP1 and truncated polypeptides of VP1.3. To study the biological activities of EV71 structural protein VP1 truncated polypeptides of VP1.Methods1. The target DNA fragments of truncated VP1 were got by reviewing the related literatures and sequence alignment.2. The primers of the DNA fragments VP1 and the truncated VP1 polypeptides (VP179～432) VP1436～864,VP1436～735,VP1565～864) were designed and amplified based on the full-length genome of EV71 SDLY107 by polymerase chain reaction (PCR).3. The recombinant plasmid were obtained by linking the target DNA fragments with the prokaryotic expression vector pET-49b(+) after double digestion.4. The recombinant vectors into the Escherichia coli BL21 (DE3) were transformed and grown in the volume of 5 ml of Luria broth medium (LB:Tryptone 10 g/L, yeast extract 5 g/L and sodium chloride 10 g/L) with 30 μg/ml kanamycin at the temperature of 37 ℃ and the vibrating frequency of 170 r/min overnight till the next day. Then the above cultures were moved to a volume of 200 ml of LB medium containing 30μl kanamycin at the temperature of 37 ℃ and the vibrating frequency of 170 r/min until the A of 0.8 at 600 nm was reached. The isopropyl-β-D-Thiogalactoside (IPTG) was added to the LB medium to a final concentration of 0.33 mmol/L to induce the expression of the target proteins post 4～5 h. The Western blot operation was conducted to detect the expression of structural protein VP1 and the truncated VP1 proteins. The proteins of the bacteria were transferred onto nitrocellulose membranes and incubated overnight at 4 ℃ after adding the mouse-EV71 polyclonal antibody. Then the HRP-conjugated goat anti-mouse IgG was added to the membranes and incubated for 30 min. The DAB kit was used for colour development to test the expression of the proteins finally.5. The structural protein VP1 and the truncated VP1 proteins harvested from the BL21 (DE3) were purified using Glutathione-Triethyleneglycocyl-Sepharose 6B.6. The detection of the reactivity of the fusion protein and EV71 polyclonal antibody by Western blot assay:Each purified GST-tagged fusion protein (the structural protein VP1 and the truncated VP1 proteins) was transferred to the nitrocellulose membranes and were incubated overnight at the temperature of 4 ℃. Then the membranes were rinsed three times with TBS with 0.01% Tween-20 and incubated in HRP-conjugated goat anti-mouse IgG for 30 min. The colour development was implemented with concentrated DAB kit to finally test whether the target proteins can be combined with the EV71 immunized sera obtained from mouse.7. The detection of the reactivity of the fusion protein and EV71 polyclonal antibody by ELISA assay:The purified target proteins were coated in the microtiter plates with phosphate buffer. The coated microtiter plates were placed in 4 ℃ overnight and blocked with 5% skim milk in PBS. The microtiter plates were incubated with EV71 immunized sera obtained from mouse for 2 h and the HRP conjugated goat anti-mouse IgG for 1 h. The reaction was developed by Tetramethylbenzidine (TMB) substrate. The optical densities were determined at 450 nm to test whether the target proteins can be combined with the EV71 immunized sera obtained from mouse.8. The purified VP1 and the truncated VP1 proteins were emulsified with Freund’s complete and incomplete adjuvant. Female BALB/c mice were intraperitoneally injected with emulsion proteins. The immune sera were collected on days between 7-10 d after the last immunization.9. The detection of the reactivity of the polyclonal antibody of the fusion protein and EV71 virus protein by ELISA assay:The proteins of EV71 were transferred to nitrocellulose membranes. The membranes were incubated in the sera of step 8 overnight at the temperature of 4 ℃. Then the membranes were incubated in the HRP-conjugated goat anti-mouse IgG for 30 min and coloration by concentrated DAB kit to test whether the EV71 could connect with the above harvested sera.Results1. The prokaryotic expression vector pET-49b(+) containing the structural protein VP1 and the truncated VP1 proteins were constructed. The target proteins can be expressed and detected in the Escherichia coli BL21 (DE3). The expression of the target proteins reaches the highest level when induced for 4 to 5 h.2. The target proteins were purified and they could react with EV71 polyclonal antibody specifically according to Western blot and ELISA assay.3. The purified target proteins were used to immune the female BALB/c mice and the sera were harvested after immunization for 4 times. The harvested sera could react with the EV71 protein specifically according to the Western bolt and ELISA assay.Conclusions1. The constructed prokaryotic expression vector could express the structural protein VP1 and truncated VP1 proteins in the Escherichia coli BL21 (DE3) in high level of expression quantity successfully.2. The structural protein VP1 and the truncated VP1 proteins showed good biological activity tested by Western blot and ELISA...