Expression And Assembly Of Foot-and-mouth Disease Virus-like Particles In Pichia Pastoris

Foot-and-mouth disease(FMD)is highly contagious and has a high incidence rate,which affects the development of animal husbandry and causes huge economic losses.At present,the inactivated whole virus vaccine is mainly used to prevent and control the disease.But,the inactivated vaccine needs to use live viruses in preparation processes,and there are certain defects,such as immunized animals.Because the inactivation of the virus can not completely cause the hidden infection or persistent virus carrying in the animal body,and even the inactivated vaccine is activated and mutated in the animal body,it can lead to the new epidemic situation of the new strain,so it is necessary to further study and develop a safe and efficient new vaccine.Compared with other vaccine forms,VLPs vaccines are relatively flexible,do not contain nucleic acids,have more conformational epitopes than natural viruses,and are more immunogenic,which is an ideal and safer form of vaccine.In this experiment,the chicken embryo domesticated attenuated strain was cloned O/Akesu/58 CE39A,and adapted and passaged continuously on well-growing BHK-21 cells.By observing the cytopathic condition,the virulence of each generation of the adapted strain was determined and picked out.RNA of the choice was extracted,reverse-transcribed cDNA,and primers for amplification of foot-and-mouth disease structural proteins VP0,VP1,VP3 genes were designed,the target genes were amplified,sequenced,and its genetic evolution relationship was analyzed.The results showed that the VP0,VP1 and VP3 genes were amplified out after transcription,and the sequence homology of VP1 gene was analyzed.It indicated that the chicken embryo domesticated attenuated strain clone O/Akesu/58 CE39A still belongs to the Middle East-South Asia topology(ME-S A).The initial vector used in this experiment is the yeast expression vector pGAPZaA,which was inserted into partial 26S rRNA genes(rDNA)of Pichia pastoris X33 strain to construct transfer vector pGAPZaAM.The plasmid included EGFP gene and the pGAPZaAM vector were digested separately to construct the expression plasmid pGAPZ?AM-EGFP.The plasmid was transformed linearly into Pichia pastoris X33 competent cells,and the recombinant strain was green under the fluorescence microscope and designated as X33-EGFP.The transfer plasmid and an EGFP recombinant strain were constructed to identify whether the expression vector pGAPZaAM can express a foreign gene.The results showed that the recombinant strain was green under the fluorescence microscope,and the EGFP gene fragment was amplified out by PCR in strain X33-EGFP.The VP0,VP1,and VP3 gene fragments were inserted between the EcoRI and NotI sites of the multiple clonal sites(MCS)of the pGAPZaAM vector to construct the transfer plasmids pGAPZ?AM-VP0,pGAPZ?AM-VP1,and pGAPZ?AM-VP3.The three plasmids were mixed in equal concentration,linearized by restriction endonuclease XmnI,and electrotransformed into Pichia pastoris X33 strain,and then screened by Zeocin(bleomycin)in YPD plates.Monoclones selected from high concentrations of Zeocin were cultured in YPD plates.The recombinant yeast was screened by PCR,SDS-PAGE,Western-bloting and electron microscopy.The results showed that the copy number of VP0,VP1,VP3 was not all 1:1:1 in the recombinant strain,the target protein was detected at 30 KD and 25 KD by SDS page and Western-blot,and the VLPs,with the size of 23-27 nm was observed by electron microscope.The specific antibody against foot-and-mouth disease could be obtained up to 1:16 in immunized mice.The above results suggest that recombinant Pichia pastoris can express and assemble foot-and-mouth disease VLPs,which is expected to provide an experimental basis for the study of foot-and-mouth disease genetic engineering vaccine.