Study On Virus-like Particle Vaccine Of Type Asia 1 Foot-and-mouth Disease Viruses

Foot and mouth disease(FMD)caused by Foot-and-Mouth Disease Virus(FMDV),is the most contagious disease of cloven-hoofed animals and has a great potential for threatening the development of animal husbandry.It is one of the OIE-Listed diseases,infections and infestations in force.At present,many countries,including China have adopted vaccination measures to control FMD.During the past years,the attenuated and inactivated vaccines played an important role in controlling FMD.However,the attenuated vaccine now was discontinued because of its risk of virulence returning in field.At present,most of the inactivated vaccines used,but there are still great biosafety risks in the production process of culture and inactivation of the virus and causing FMD outbreaks due to incomplete inactivation.Therefore,it is necessary to develop a safe and effective new vaccine for FMD.With the rapid development of immunology and genetic engineering technology,the main functional structure protein genes of FMDV were expressed,and virus-like particles(VLPs)were assembled in vitro.In this study,the prokaryotic expression system was used to clone the VP0,VP1 and VP3 structural protein genes of type Asia 1 FMDV into the expression vector,and the recombinant bacteria were constructed to induce the VP0,VP1 and VP3 proteins of type Asia 1 FMDV to be expressed,and then assembled into type Asia 1 FMDV VLPs in vitro.Novel vaccine based on VLPs was developed to obtain good immune effect.1.Construction of expression vectors: The Smt3 gene was amplified from total DAN of Saccharomyces cerevisiae and inserted into a pET-28 a vector to get plasmid designated as pSMK.The gene coding the kanamycin in pSMK vector was replaced by the gene of ampicillin,the recombinant vector was named pSMA.The genes,VP0,VP3 and VP1 from FMDV type Asia 1(Asia 1/JSL/06 strain),were cloned into pSMK,pSMA and pSMK,respectively.The resultant plasmids were designated as pSMK/VP0,pSMA/VP3 and pSMK/VP1,and the gene sequence containing the T7 promoter and VP1 DNA fragment was amplified using PCR from pSMK/VP1 template,then inserted into pSMK/VP0 vector constructed the recombinant vector with dual genes was constructed as pSMK/VP0-VP1.Both of pSMA/VP3 and pSMK/VP0-VP1,which was co-transformed into the host,BL21(DE3)strain named BL21(pSMK/VP0/VP1/VP3).2.Expression and purification of FMDV type Asia 1 VLPs: An overnight culture of BL21(pSMK/VP0/VP1/VP3)cells was cultured in fresh Luria-Bertani(LB)medium(plus ampicillin)until cells reached mid-log growth.The culture was induced by the addition of IPTG.The recombinant fusion protein,VP0/VP1/VP3,observed in highly water-soluble by SDS-PAGE,and the target protein has better reactionogenicity by Western blot assay.Cleavage of the SUMO tag from the fusion protein is to yield authentic VLPs.The VLPs was viewed using TEM show that it can be assemble into Cap-like particles similar with 75 S of native FMDV capsid,which is FMDV type Aisa1 VLPs.3.VLPs vaccines safety and immunogenicity:Type Asia 1 FMD VLPs containing different contents were used as antigens and mixed together with adjuvant 206(1:1)to produce VLPs vaccines.Guinea pigs,pigs and cattle were vaccinated to investigate the safety and immunogenicity of type Asia 1 FMD VLPs vaccine.During the 10 days immunization period,all the vaccinated animals show good mental state and no obvious clinical adverse reactions which confirmed that the type Asia 1 FMD VLPs as antigens were safe for animals.The antibody against type Asia 1 FMDV in sera from the experimental animals was detected by liquid phase blocking ELSIA and the results revealed that the antibody of pigs and cattle reached a peak at 21-28 days after immunization and 87.5%(14/16)of the animals have produced specific antibody against type Asia 1 FMDV.The challenge test showed that the protection ratios for pigs and cattle were 15/16 and 14/16 respectively,which represented that the VLPs could induce immune response in animals.4.Determination of the appropriate antigen contents in VLPs vaccine: Prepare the vaccine with 200?g,100?g,50?g and 25?g of Asia 1 type FMD VLPs antigens per dose respectively,to immunize pigs and cattle.Cattle for 21 days and pigs for 28 days after vacciantion were challenged with Asia 1/JSL/GSZY/06.It was found that the protection rates and the immunization qualification rates of the first three vaccines(containing 200?g?100?g?50?g antigens)are all high than 75% and 70% respectively.The protection rates and the immunization qualification rates of the animals immunized with 25?g content is low than 75% and 70% respectively.According to the standards of OIE and China's Ministry of Agriculture and Rural Affairs,the antigen contents of type Asia 1 FMD VLPs vaccines was determined to be 100?g per dose to guarantee the quality and immune effect.5.Vaccine Efficacy Test: In order to evaluate the VLPs vaccine efficacy,total 48 Cattle and 48 pigs vaccinated with 3 batches of type Asia 1 FMDV VLPs vaccines produced at laboratory.The animals challenged with Asia 1/JSL/GSZY/06 strain on 21 dpv for cattle and on 28 dpv for pigs.The vaccine protection rate for both cattle and pigs are all 87.5%(42/48),which meet the FMD vaccine quality standard of OIE and China.6.Immunity duration of VLPs vaccine: Blood samples detected using LPB-ELISA at 1,2,3,4,5,6 and 7 months from cattle and pigs,which vaccinated with 3 batches of type Asia 1 FMDV VLPS vaccines produced at laboratory.At 6 and 7 months after vaccination,the animals challenged with Asia 1/JSL/GSZY/06 strain to evaluate the antibody level and immunity duration.The levels of FMDV Asia 1 antibody in cattle and pigs are basically the same.The antibody level remained high during 1-3 months after vaccination,and then gradually decreased.At 6 month after vaccination,the qualified rate of vaccination was nearly 70% and it is lower than 70% at 7 months after vaccination.Animals challenged tests show that the protection rates of cattle and pigs are higher than 75% at 6 month after vaccination,while only one batch vaccinated animal protection rate is reached 75%.Based on the above data,the vaccine immunity duration is determined as about 6 months.