猪流行性腹泻病毒HN02分离株S1蛋白的真核表达及其活性鉴定OA

In order to prepare the recombinant S1 protein of Porcine epidemic diarrhea virus(PEDV)and evaluate its antigenic activity,the signal peptide and structure of the S1 protein were predicted;a eukaryotic expression plasmid was constructed;and the protein was expressed,purified,and identified using HEK293F cells.Western-blot was used to detect cell samples at 24,48,72,and 96 h post-transfection to determine the optimal harvest time for the S1 protein.After treatment of the S1 protein with PNGase F glycosidase,deglycosylation analysis was performed using SDS-PAGE and Western-blot;bio-layer interferometry(BLI)and ELISA were used to evaluate the affinity of the S1 protein with the monoclonal antibody and its reactivity with PEDV-positive serum.The results showed that specific erythema appeared in HEK293T cells transfected with S1 plasmid,indicating normal expression of S1 protein.The S1 protein was secreted and expressed in HEK293F cells through its own signal peptide.The expression level reached its peak at 72 h after transfection;thus,72 h post-transfection was selected as the optimal time for sample collection.The washing condition for S1 protein was 10 mmol/L imidazole,and the elution condition was 100 mmol/L imidazole.The purity of the purified S1 protein exceeded 90%.After treating the S1 protein with PNGase F,a glycosylation enzyme,the molecular weight decreased,indicating that the HEK293F system could glycosylate the S1 protein.The dissociation constant(KD)value of the S1 protein with PEDV monoclonal antibody(mAb)was 55.6 nmol/L,indicating high affinity.ELISA titer of S1 protein for detecting PEDV-positive serum was over 1∶25 600,suggesting that the S1 protein possessed good antigenic activity and could be utilized for the detection of PEDV antibodies.It was demonstrated that the S1 protein with high purity,high expression level,and good glycosylation modification was secreted and expressed in HEK293F cells,and it could react with PEDV mAb and positive serum with high affinity.