High-Level Secretory Expression Of Highly Pathogenic H5N1 Avian Influenza Virus HA1 Protein In Pichia Pastoris And Its Study

The recent outbreaks of highly pathogenic H5N1 influenza A virus not only cause a large amount of economic loss to farms but also endanger people's life,A broad spectrum vaccine will be the most effective way to limit the spread of highly pathogenic H5N1 influenza virues.P.pastoris as a cellular host for expression of recombinant proteins is easier to genetically manipulate and culture than mammalian cells and can be grown to high cell densities.Equally important,P.pastoris is also a eukar yote,and thereby provides the potential for producing soluble,correctly folded recom binant proteins that have undergone all the post-translational modifications required for functionality.Here we report the high-level secretory expression of active recombinant HA1 protein in p.pastoris and the recombinant HA1 proteins was proved to be a good candidate vaccine.A series of important epitopes were predicted and verified with the help of bioinformatics.The crystal of 13D4 Fab-rHA1 co mplex was obtained,this provided a probability to elucidate the conservative epitope of H5N1 virus.The expression plasmid pPIC9K-HA1 was transformed into P.pastoris host cell GS115,and the recombinant engineering strains for secretory expression were constructed and screened.The positive strains containing multiple gene dosages were screened out by G418-YPD plates,and the results showed that the higher gene dosages had the effect of increasing the amount of rHA1 expression.Then the influence of different factors on biomass and recombinant HA1 protein production during induction phase in shake flask were studied.The optimized cultivation conditions were original pH at 5.4,concentration of methanol at 1.0%and cultivation temperature at 25℃.Based on the research in shake flask,the recombinant HA1 protein was produced by high-cell density fermentation,after optimizing the fermentation process,the rHA1 yield in 10L-fermentor(about 120mg/L) was 10 times higher than that in shake flask(about 11mg/L),with the final OD₆₀₀ at about 320.The expression level of rHA1 could improve 5 times by deleting N-terminal 48aa.The supernatant of the fermentation culture was concentrated and buffer-exchanged by crossflow filtration,and then purified by ion-exchange chromatography.The purified protein recovery was about 24%.After detected by a panel of 93 anti-H5 HA monoclonal antibodies(mAbs),the recombinant HA1 was proved to have good antigenicity.Mice was immuned with recombinant HA1 protein,after 5 weeks,the titers of anti-rHA1 serum reached 1.2×10⁴ and the anti-rHA1 serum had high Hemagglutination inhibition(HI) titers to YU22 and 2439 avian virus but relatively low Hemagglutination inhibition titers to Yu324,213 avian virus.Anti-rHA1 serum was tested against 4 neutralization monoclonal antibodies(13D4,8H5,8G9,10F7) in blocking assay,anti-rHA1 serum could block the reaction between neutralization monoclonal antibodies and YU22 virus.The deletion of N-terminal 48aa won't lower the biologic activity of rHA1.This suggested that the recombinant HA1 protein produced by P.pastoris was a good candidate vaccine.In the former study,our research group had predicted many important epitopes of HA1 by bioinformatics.In this study,we did site-directed mutagenesis of these specific amino acids,and compared the biological activity bewteen normal rHA1 and mutated rHA1,finally we found that Glu¹¹²,Lys¹¹³,Ile¹¹⁴ was important for the correct folding of HA1,Pro¹¹⁸ and Tyr¹³⁷ were the key amino acids of epitopes of 1A6 and 13H8 respectively,Asn¹⁶⁵ was an important amino acid for HA1 to react with 13D4, an a mutaion of a loop from aa123 to aa132 would greatly lower the biological activity of rHA1.After deleting 48aa from N-terminal,the expression level of rHA1 mutant was improved and was up to 520mg/L,and the rHA1 mutant remained the original biologic activity of HA1.At last,we studied the crystallization of 13D4 Fab-rHA1 complex,and got some cuboid crystals of 13D4 Fab-rHA1 complex with the size 0.2mm×0.2mm×0.05mm. This lay a foundation for the analysis of 13D4 Fab-rHA1 structure by X-ray.