The Immunogenicity Of RBD Protein Expressed By Pichia Pastoris Is Affected By Its N-glycosylation Modification

N-glycosylation,one of the most common post-translational modifications in eukaryotes,mainly occurs in the endoplasmic reticulum and Golgi apparatus.Glycosylation has essential effects on protein folding,signal transduction,fertilization and embryo development,cell proliferation and tissue formation,and other life processes.The capsid protein on the surface of the virus also has a ubiquitous glycosylation modification.The viral glycosylation modification antigen can mask the antigen epitope and inhibit the host's immune response so that the virus can escape from immunological recognization and clearance.It has been reported that the Spike protein of SARS-Co V-2 contains 22 N-glycosylation modification sites,and RBD contains two N-glycosylation sites,in which the three smallest mannose modification has the most miner shielding effect on S protein,and the nine largest mannose modification in mammals has the strongest shielding effect,ranging from 29%to 47%.The difference in glycosylation may affect the recognition and internalization of S protein by antigen-presenting cells in the immune system and the recognition of neutralizing antibodies,Ultimately affecting glycoprotein vaccines'immunogenicity.Based on the different glycosylated Pichia strains constructed in our laboratory and the RBD glycoprotein containing two N-glycosylation modification sites used as the model protein,we aimed to study the effects of different N-glycosylated glycosyl structures on the production of binding antibodies and neutralizing antibodies of RBD subunit vaccines to find a sugar type which improves the subunit vaccines'immunogenicity.Main research results are as follows:1.Different glycosylations modified RBD proteins were prepared by wild-type Pichia pastoris X33 and glycosyl-engineered Pichia pastoris GJK05.The glycoproteins expressed by wild-type Pichia pastoris X33 all have heterogeneous excessive mannosylation,and the bands are diffuse.To study the effect of excessive mannosylation on the immunogenicity of RBD glycoproteins,a purification scheme was initially established.The study found that the total RBD protein captured by cation exchange chromatography can be further divided into different molecular weights by hydrophobic interaction chromatography:high molecular weight RBD protein modified with excessive mannose(h-MAN),and medium molecular RBD protein modified with excessive mannose(m-MAN),low molecular weight RBD protein(L-MAN)modified with excessive mannose.It is further found that anion exchange chromatography can divide the over-mannose RBD protein of medium molec?Lar weight into negatively charged glycoform RBD protein(m MAN-P)and neutral sugar RBD protein(m MAN-n).To further study the effect of excessive mannose modification,the laboratory-built glycoengineered yeast GJK05 with excessive mannose deficiency was used to induce expression and purification to obtain low molecular weight oligomannose RBD protein(o-MAN).The Previous report reveals that?-mannose can effectively mediate the uptake of antigen by DC.In this case,we constructed a?-mannosyltransferase expression plasmid and a?-mannose-type expression strain on the basis of GJK05 bacteria,and purified?-mannose-type RBD glycoprotein(o MAN-?).The complex glycoform RBD(complex)protein expressed by the glycosyl engineered Pichia yeast constructed in our laboratory has a good immune protection effect.We use this as a control to verify the immunogenicity of other glycoform RBD proteins.The results of SDS-PAGE and western blot showed that the molecular weight of the total RBD protein expressed by X33 was between 40k D-60k D,in a diffuse state.After dividing into different components according to the nature of the RBD glycoprotein,the band uniformity was improved,but still in a diffuse state.It is consistent with the heterogeneous glycosylation modification of X33 expressing glycoprotein.The molecular weight of the RBD protein expressed by the over-mannose-deficient strain was significantly smaller than the RBD protein expressed by X33.The molec?Lar weight of RBD proteins of different glycoforms was reduced significantly after PNGase F digestion,and the molecular weight was consistent with the theoretical molecular weight,indicating that the difference in molecular weight displayed by SDS-PAGE was caused by N-glycosylation modification.2.The over-glycosylated medium molecular weight RBD protein(m MAN-P)with negatively charged sugars has a better immune effect.The purified RBD protein was immunized with 5?g protein plus 100?g Al(OH)₃ adjuvant on day 0 and day 14 to immunize Bal B/C mice,and blood was collected from the tail vein before and after the first immunization and 14 days after the second immunization.Then use ELISA to detect its binding antibody titer and neutralizing antibody titer,and further distinguish the types of induced Ig G antibodies.First,complex RBD,m MAN-P RBD and deglycosylation RBD proteins were used as detection antigens,and ELISA was used to detect the binding antibody titer.The res?Lts showed that the binding antibody titers induced by m MAN-P type RBD protein were 12.5 times(P=0.0001),8.3 times(P=0.0020),and 9.8 times(P=0.0013)higher than complex glycoform RBD protein,respectively,with significant differences.It also shows that the antigen used for detection does not affect the detection of bound antibody titer.Subsequently,SARS-Co V-2 pseudovirus packaged with 293T cells was used to detect the neutralizing antibody titer.The results showed that the arithmetic mean of neutralizing antibody titer induced by m MAN-P type RBD was 4.6times higher than that of complex glycoform RBD(P=0.0020),the difference was significant.We further analyze the Ig G antibody subtypes induced by RBD proteins of different glycotypes.The results showed that Bal B/C immunized with RBD protein plus aluminum hydroxide mainly induced Ig G1 antibodies.And except for the RBD expressed by GJK05-arm2 which can strongly induce the production of Ig G2a subtype antibodies,the ability of other glycotype RBD proteins to induce different subtypes of Ig G is consistent with total Ig G.In summary,we successfully obtained h MAN,m MAN-P,m MAN-n,o MAN and o MAN-?RBD glycoproteins,using wild-type Pichia pastoris X33 and excessive mannose-deficient strains.Compared with the complex glycoform RBD commonly used in the production of protein vaccines,the RBD protein(m MAN-P type RBD protein)is expressed by X33,which is excessively mannose and has a negative charge at can induce stronger binding antibody titer and neutralizing antibody titer.This may have an impact on the optimization of the production of new coronavirus vaccines and the design of subunit vaccines based on Pichia pastoris...