Secretion Expression Of Hepatitis B Core Antigen Particles Harboring HEV Epitope In Pichia Pastoris

The Pichia pastoris expression system has become increasing popular in recent years.As a eukaryotic expression system,it can perform many post-translational modifications required for functionality,such as processing of signal sequences, folding,disulfide bond formation,certain types of lipid addition and O- and N-linked glycosylation.Furthermore,Pichia pastoris can extracellularly express heterologous proteins.Because this yeast secretes only low levels of endogenous proteins, compared with intracellular expression,extracellular expression has nothing with release of cell contents and separation of cell debris during cell disruption and the secreted proteins can easily purified.Hepatitis E is a global infectious disease caused by hepatitis E virus(HEV) and it's endemic in Asia and Africa and Mexico.The viral genome consists of three open reading frames(ORFs) and the second open reading frame(ORF2) encodes a peptide of 660 amino acids,which is the structural protein of the virus.Some researches show that the amino acids 423-438 of ORF2,recognized by the monoclonal antibody 12A10,play an important role in binding between the virus and host cells.The hepatitis B core antigen,which is an excellent immuno-carrier,can self-assemble in particles in both eukaryotic and prokaryotic expression systems.The heterologous epitopes can be inserted into the major immunodominant region of the protein,which still can assemble into particles,and the epitopes are well displayed on the surface of the particles.In our study,the gene encoding HBcAg fused with 12A10 epitope was introduced in Pichia pastoris host cells and the recombinant engineering strains for secretory expression were constructed and screened.Then the culture conditions were optimized and the purified protein was analyzed.The 12A10 epitope in the fusion protein was proved to have good immunogenicity.The results of our study may shed light on the secretion expression of large-size particles in P.pasloris and show insight into the rational design of the vaccine carrier.Firstly,the hepatitis B virus core gene fused with the HEV epitope gene,which might involve distinct receptor-binding sites,was cloned into the expression vector pPIC9K to construct the recombinant expression vector pPIC9k-HBc149-12A10. After linearized by restriction endonuclease Sac I,the recombinant expression vector was transformed into Pichia pastoris strain GS115 by electroporation.The transformants with high resistance to G418 were cultivated and induced by methanol. SDS-PAGE analysis showed that,compared with that of GS115/pPIC9k,the culture supernatant appeared a special protein-band of about 22KD,which could specifically reacted with 12A10 mab in the Westen Blot.Secondly,the recombinant engineering strain was cultivated respectively in 3 groups of culture medium:MGY/MM,BMG/BMM and BMGY/BMMY.Indirect ELISA test showed the BMGY/BMMY medium was obviously better than the other two.On the basis of BMGY/BMMY medium,the effects of original pH value, concentration of methanol and cultivation temperature were studied by single factor experiment.Then by using orthogonal experiment design,the optimum cultivation condition was determined as follows:riginal pH value at 7.0,concentration of methanol at 1.0%and cultivation temperature at 25℃.The supernatant was concentrated and buffer-exchanged by crossflow filtration,and then was purified by hydrophobic interaction chromatography.Finally,CsCl gradient centrifugation analysis indicated the density of the recombinant protein was 1.32g/ml.TEM observation indicated that the fusion protein could self-assemble into particles,which was approximately 30nm in diameter..Mice were immunized with the recombinant paticles,and 8 weeks later the titers of the mice antiserum reached 1.6×10~5.The 12A10 antibody level in the mice antiserum much higher than that in the serum from mice immunized with the p239 protein carrying 12A10 epitope,which suggested that non-immunodominant epitope was better presented in the recombinant particles.Our study could be seen as a reference to the secreted expression of recombinant virus particles by P.pastoris and an example of the vaccine carrier;it was also a new solution to the problem of difficult purification in the yeast intracellular expression.