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目的对重组戊型肝炎疫苗(recombinant hepatitis E virus,rHEV)抗原P179表征进行分析。方法取3批HPLC及电泳纯度均为100%的rHEV抗原P179原液,采用SDS-PAGE法检测相对分子质量及二聚体含量;OPA-FMOC全自动柱前衍生-Amino Quant氨基酸分析法测定氨基酸组分;电喷雾-四极杆-飞行时间串联质谱(electrospray ionizationquadrupole time-of-flight-mass spectrometry,ESI-Q-TOF2-MS)法分析C-末端、Edman降解法测定N-末端氨基酸序列;HPLC法进行肽图分析;毛细管等电聚焦电泳法测定等电点;圆二色光谱仪分析其二级结构;透射电镜负染法观察蛋白颗粒状态;双抗体夹心ELISA法检测抗原活性。结果 3批rHEV抗原P179相对分子质量在17 500~21 300之间;二聚体占总蛋白的85%以上;氨基酸组分、C-末端氨基酸序列、N-末端氨基酸序列、肽图、等电点等均与预期相符;3批rHEV抗原P179二级结构中,α螺旋4.8%~5.6%,β折叠34.8%~36.4%,β转角20.8%~22.2%及无规卷曲37.7%~38.0%;透射电镜观察可见大小均一,粒径约20 nm的蛋白颗粒;抗原活性比值在5.0×105~2.0×10~6CCU/mg范围内。结论 3批r HEV抗原P179结构特征均一,与设计相符,为该疫苗的产业化奠定了基础。
Objective To analyze the characterization of P179 antigen of recombinant hepatitis E virus (rHEV). Methods Three batches of rHEV antigen P179 with HPLC and electrophoresis purity of 100% were used to detect the relative molecular mass and dimer content by SDS-PAGE, and the amino acid group was determined by OPA-FMOC automated pre-column derivatization-Amino Quant amino acid analysis The C-terminus was analyzed by electrospray ionization quadrupole-time of flight mass spectrometry (ESI-Q-TOF2-MS) and the N-terminal amino acid sequence was determined by Edman degradation. HPLC Method was used to analyze the peptide structure; the isoelectric focusing point was determined by capillary isoelectric focusing electrophoresis; the secondary structure was analyzed by circular dichroism spectroscopy; the state of protein granules was observed by negative staining with transmission electron microscopy; the antigen activity was detected by double antibody sandwich ELISA. Results The relative molecular mass of the three batches of rHEV antigen P179 was between 17 500 and 21 300; the dimer accounted for more than 85% of the total protein; the amino acid composition, C-terminal amino acid sequence, N-terminal amino acid sequence, peptide map, The results showed that the secondary structure of P179 rHEV antigen was 4.8% -5.6% in α-helix, 34.8% -36.4% in β-sheet, 20.8% -22.2% in β-sheet and 37.7% -38.0% in random coil. Transmission electron microscopy showed uniform size and particle size of about 20 nm protein particles; antigen activity ratio in the 5.0 × 105 ~ 2.0 × 10 ~ 6CCU / mg range. Conclusion The structural characteristics of three batches of r HEV antigen P179 are uniform and consistent with the design, laying a solid foundation for the industrialization of the vaccine.