Identification Of Glycosylation Sites In GP5 Protein Of Two PRRSV Strains And Study On Immune Responses Induced By Mutants Vaccine

Porcine reproductive and respiratory syndrome(PRRS)is a viral infectious disease caused by porcine reproductive and respiratory syndrome virus,which can lead to abortion of pregnant sows,respiratory symptoms of piglets and high mortality,commonly known as blue ear disease.Since its discovery,people have realized that the clinical symptoms of the disease are very different among individuals.PRRSV does not produce special clinical symptoms.Its clinical symptoms reflect the virulence of PRRS virus,the age and immune status of pigs and the existence of concurrent infection.Overall,the most common clinical symptoms of PRRSV infection include anorexia,fever,lethargy and respiratory diseases.In the breeding population,clinical symptoms may include infertility,abortion,premature delivery,stillbirth,increased number of weak pigs,and acute diarrhea in newborn piglets.The disease was first found in the Midwest of the United States in 1987.China first appeared in Taiwan in 1991 and broke out and spread in the mainland in 1996.PRRSV infection causes immunosuppression,which leads to immune failure of other vaccines in pigs and frequent mixed infection.In recent years,the clinical prevalence of PRRSV has become more complex due to the variability of PRRSV gene and the emergence of new epidemic strains such as PRRSV NADC30 like / NADC34 like.GP5 protein is encoded by ORF5 and is the main structural protein of PRRSV.Compared with other structural and non structural proteins,GP5 has good immunogenicity,which is mainly reflected in the neutralization effect of GP5 induced antibody on PRRSV.Studies have shown that GP5 protein has two B-cell antigen epitopes,among which the antigen epitope in the downstream produces neutralizing antibody,while the one in the upstream belongs to bait epitope.It is speculated that the bait epitope in the upstream produces a large number of non neutralizing antibodies in the early stage of PRRSV infection,inhibits the neutralizing epitope in the downstream B-cell,induces the body to produce neutralizing antibody,and leads PRRSV to escape the humoral immune response of the body.At present,some studies have confirmed that PRRSV relies on the glycosylation modification of its envelope protein to escape the host immune response,while removing the N-glycosylation modification near the neutralization epitope of PRRSV GP5 can make the virus more easily neutralized by antibodies,and the mutant virus carrying GP5 glycosylation deletion causes stronger neutralization antibody response than wild-type virus.Therefore,it is speculated that the N-glycosylation around the neutralization epitope of GP5 protein forms glycan shielding effect and promotes the immune escape of PRRSV.In this study,the GP5 protein N35 and N51 sites of PRRSV epidemic strain NADC30 like CHsx1401 and classical strain VR2332 were mutated by overlap PCR.The mutation direction was basic amino acid arginine R(AGA).The mutants were connected to the eukaryotic expression vector p CAGGS EGFP to construct the recombinant expression vectors:p CAGGS-CHsx1401-ORF5,p CAGGS-VR2332-ORF5,p CAGGS-CHsx1401-N35-AGA,p CAGGS-VR2332-N35-AGA,p CAGGS-CHsx1401-N51-AGA,p CAGGS-VR2332-N51-AG A,in order to eliminate the possibility that glycosylation may be affected by the acidity and alkalinity of mutant amino acids,based on the preparation of the above recombinant plasmid,another acidic amino acid mutation vector at N35 and N51 sites was prepared.The mutation direction is acidic amino acid aspartic acid D(GAC): p CAGGS-CHsx1401-N35-GAC,p CAGGS-VR2332-N35-GAC,p CAGGS-CHsx1401-N51-GAC,p CAGGS-VR2332-N51-GAC.The corresponding eukaryotic expression plasmids were transfected into 293 T cells by liposome transfection method.The expression of GP5 protein in the cells was identified by Western blot.The expression proteins of p CAGGS-CHsx1401-ORF5 and p CAGGS-VR2332-ORF5 were treated with two endoglycosidases.The GP5 protein bands of the two untreated strains were located at or above 50 KDa.After treatment,the bands were moved down to or below 50 KDa,which proved that many bands had N-glycosylation modification,It is speculated that the upper band is high-level glycosylated GP5 protein and the lower band is low-level glycosylated GP5 protein.In order to study the effect of N35 and N51 site mutations on the glycosylation level of GP5,the protein produced by the corresponding recombinant eukaryotic expression plasmid was identified by Western blot.The results showed that after the acid and alkaline mutations of N35 and N51 amino acids,a new protein band appeared between the high-level glycosylation band(Upper band)and the low-level glycosylation band(lower band),which proved that the mutations of N35 and N51 sites changed the structure and morphology of GP5 protein.The gray value analysis of each band showed that the total amount of GP5 protein expressed by N35 mutants(p CAGGS-CHsx1401-N35-AGA,p CAGGS-VR2332-N35-AGA)after transfection into 293 T cells was slightly higher than that in the original non mutant group,but there was no significant difference;The total amount of GP5 protein expressed by the N51 mutant(p CAGGS-CHsx1401-N51-AGA,p CAGGS-VR2332-N51-AGA)was significantly lower than that in the original non mutant group.The comparison of high-level glycosylated GP5(upper band)of Western blots in each group showed that the amount of high-level glycosylated protein expressed by N35 and N51GP5 mutants of the two strains was significantly or very significantly lower than that of the original p CAGGS-ORF5,indicating that the mutation of glycosylated site significantly reduced the expression level of high-level glycosylated GP5(upper band).The comparison of low-level glycosylated GP5(upper band)of Western blots in each group showed that the amount of low-level glycosylated protein expressed by the N51 GP5 mutant of the two strains was significantly or very significantly lower than that of the original p CAGGS-ORF5.The amount of low-level glycosylated protein expressed by N35 GP5 mutant was not different from that of the original p CAGGS-ORF5,the total amount of GP5 protein expressed by N35 mutant was not significantly different from that of the original p CAGGS-ORF5,and the expression of high-level glycosylated protein was significantly lower than that of the original p CAGGS-ORF5.We speculate that the medium glycosylation level protein expressed by N35mutant(the middle band appears after mutation)is caused by the deglycosylation and downward migration of high glycosylation level protein(the upper band).Studies have shown that high levels of glycosylation will cover antigenic epitopes,including neutralizing epitopes.On the basis of no significant difference in the total protein expression between the mutants at N35 site of the two strains,the expression of high-level glycosylated GP5 was significantly reduced.Therefore,the PRRSV epidemic strain NADC30 like and the original GP5 and N35 mutation of classical strain VR2332 were prepared into nucleic acid vaccine,and rabbits were immunized according to the set immune procedure.Serum was collected regularly to determine its immune expression response and evaluate its immune effect.Results on the 14 th and 28 th days,the neutralizing antibody in the serum of rabbits immunized with N35 mutation nucleic acid vaccine group was significantly higher than that in the original GP5 group,and there was a significant difference in p CAGGS-VR2332-N35-AGA group on the 28 th day.Indicating that N35 mutation recombinant nucleic acid vaccine can stimulate the body to produce stronger humoral immunity.From the above results,it can be preliminarily concluded that the single glycosylation site of mutant PRRSV GP5 N35 and N51 significantly changed the glycosylation level of GP5 protein,the expression of high-level glycosylated protein and low-level glycosylated protein,and preliminarily determined the role of this site in protein glycosylation;The N35 mutant also induced a significantly increased neutralizing antibody,suggesting that the mutation changed the glycosyl masking of neutralizing epitopes.This study accumulated data for the in-depth study of the glycosylation level and function of PRRSV structural protein,and provided ideas for the research and development of new PRRS vaccine.