Quantification Detection of Recombinant Porcine Pseudorabies Virus gD Protein by High Performance Size-Exclusion Chromatography
This study aims to establish a high performance size-exclusion chromatography(HPSEC)method for quantitative quality control of recombinant porcine pseudorabies virus(PRV)glycoprotein gD,and to evaluate its application,further extending the its use in veterinary vaccines.The gD protein standard was prepared and identified,and its stability during storage was assessed.The HPSEC method was used to quantitatively detect gD protein and identify its chromatographic peak.A standard curve was established for the HPSEC method using gD protein standard,and linearity,detection limit,accuracy,and precision were validated.The applicability of HPSEC for quantifying gD protein concentrations in cell culture media,purified samples,and emulsified samples was investigated.The gD protein quantitative results obtained by HPSEC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)were compared.The results showed that the prepared gD protein standard had a purity of>98%,was stable for 12 months,and the concentration deviation ranged from 0.01%to 2.80%.The HPSEC method exhibited excellent linearity(R2=0.999 9,n=8),a detection limit of below 5.0 μg/mL,high accuracy(93.2%-104.6%for five concentrations of gD protein standard,n=6),and good precision[relative standard deviation(RSD)of 0.2%-1.6%for five concentrations,n=6].The method showed good intra-day precision,with RSD of 1.1%-2.0%for six repeated tests per day over three days(n=6)for three batches of cell culture media.HPSEC was applicable for monitoring gD protein expression levels in cell culture media,and for detecting gD protein in purified and emulsified samples.Quantification of gD protein in 12 batches of cell culture fluid by HPSEC and SDS-PAGE showed a highly positive correlation(Rs2=0.929 8,n=12),with no significant difference between the methods in paired t-tests(Ps=0.145 7).These findings indicate that the HPSEC method for quantitative detection of PRV gD protein is suitable,reproducible,and accurate,and can be used for quality control at various stages of antigen production and vaccine process development.
high performance size-exclusion chromatography(HPSEC)porcine pseudorabies virus(PRV)gD proteinvaccinequantification
万方数据
维普
