Preparation And Immunological Activity Of Nanocellulose Conjugated O-type Foot-and-mouth Disease Virus VP1 Antigen

Foot-and-Mouth Disease(FMD)is a highly infectious,acute,febrile disease caused by Foot-and-Mouth Disease Virus(FMDV)that mainly infects cloven-hoofed animals.The high incidence rate and strong contagious nature causes serious losses to the global animal husbandry industry.To prevent and control of foot-and-mouth disease,the development of a new efficient and safely use of the vaccine becomesa significant necessary.VP1 is the most important antigenic structural protein of FMDV,which located in the capsid structure of FMDV and exposed to the surface of the virus particles.Nano-sized cellulose exhibits novel characteristics of large surface area,good biological activity,and non-toxicity,and can be used as a carrier for antigen protein delivery to enhance antigen immune effect.CBD(cellulose-binding domain),a natural structural protein has a great affinity to bind crystalline cellulose specifically.The aim of this study was to prepare a VP1 foot-and-mouth disease antigen containing a CBD protein conjugated with nanocrystal cellulose and evaluate itsimmune activity against FMD.The investigation was carried out stepwise as follows:1.Construction of recombinant plasmid p ET19b-CBD-VP1: Oligonucleotide primers were designed and synthesized,CBD DNA was amplified by PCR using the plasmid of p UC-sp-CBD-sc Fv as a template.After digestion and purification,the PCR product was inserted into plasmid p ET19b-CBM-VP1 to replace CBM DNA fragment.Five of 15 recombinants obtained from the experiment were selected for PCR,and analyzed by gel electrophoresis and DNA sequencing.The results showed that the recombinant plasmid of p ET19b-CBD-VP1 was successfully constructed.2.Prokaryotic expression of p ET19b-CBD-FMDV-VP1 and determination of optimal induction conditions: The recombinant plasmid p ET19b-CBD-FMDV-VP1 was transformed into the competent strain E.coli BL21(DE3),the commonly used for protein expression,from which the monoclonal was selected for protein expression in this study.The molecular weight of the expressed product was about 58.6 KDa analyzed by SDS-PAGE,identical to the expectation.The expression product was confirmed by Western blotting assay as the CBD-FMDV-VP1 target protein.IPTG was used to induce the protein expression,the optimal induction comditions was investigated including the concentration of IPTG,duration of action,and temperature,which was0.4 m M IPTG at 37 °C for 6 h incubation.3.Purification and renaturation of CBD-FMDV-VP1: The target protein mainly was expressed in the form of inclusion bodies from the expression host.To harvest the product of target protein,the cells were first ultrasonically disrupted,the inclusion bodies were collected by centrifugation.The inclusion bodies were then redissolved in a denaturant and the CBD-FMDV-VP1 protein was purified by nickel column affinity chromatography.The optimal imidazole concentrations of Wash Buffer and Elution Buffer were found as 4 m M and 50 m M,respectively.After purification,the denatured protein was renatured again.4.Preparation of nanocellulose: In this experiment,nano-cellulose crystal was obtained by acidic hydrolysis using softwood pulp as a raw material 64% of sulfuric acid as reagent.The hydrolysis was carried out at 45 °C for 45 min.The product was collected by centrifugation and dialyzed against deionic water unter p H kept consistant in the dialysis system.The final product was ultrasonicated and filtration through a sequence of 1.0,0.45 and 0.22 μm syringe filter.The particle zise,size distribution and zeta potential were characterized by dynamic light scattering,and electron microscope.The length of the nanoparticle was in the range of 100-300 nm,the width was in the range of 10-40 nm,the average hydrodynamic diater was 169 nm determined by Nanosizer.5.Conjugation of CBD-FMDV-VP1 protein to nanocellulose: The renatured CBD-FMDV-VP1 protein solution was mixed with nanocellulose crystal suspension to combine the two components to form a nano protein conjugate of CBD-FMDV-VP1-CNP,and the binding conditions were optimized.The binding efficiency was analyzed by SDS-PAGE and Dot Bloting.It was found that the optimal mixing ratio of nanocellulose to protein CBD-FMDV-VP1 was 10:1for 6h at 4 °C The results also showed that the obtained CBD-FMDV-VP1-CNP could efficiently bind specifically to VP1 murine m Ab.6.Preliminary study on the immunogenicity of CBD-FMDV-VP1 and CBD-FMDV-VP1-CNP: 18 male Kunming male mice aged 6-8 weeks were selected,and divided into 6 groups,which were labeled as group A,group B,group C,group D,group E and group F.The first immunization was performed by intraperitoneal injection of the CBD-FMDV-VP1 protein emulsified with Freund’s complete adjuvant into the A-E group.The injection dose of each group of A-E mice was 0 μg,25 μg,50 μg,75 μg,and 100 μg,respectively,the injection dose of first immunization for F group was 100 μg.On the 21 st day after the first immunization,each group of mice received a second immunization in the same manner as the first immunization withhalf dose of the emulsified protein.The serum from treated mices was collected and analyzed by Western blotting and ELISA..Western blotting results showed that VP1 antibody was contained in the serum of mices immunized with CBD-FMDV-VP1 and CBD-FMDV-VP1-CNP.The results of indirect ELISA analysis showed that VP1 antibody was produced in the serum of the B and E group on the 7th to 35 th days after the first wash,and the antibody level increased with time and antigen dose.The antibody titer of 28 days after immunization was 1:3200.While the antibody of VP1 was also produced in the serum of group F on the 7th-35 th days after the first wash,and the antibody level increased with time in 28 days after immunization.The VP1 antibody titer produced in the serum of group F was 1:6400.