重组戊型肝炎疫苗抗原P179表征分析  

Characterization analysis of recombinant hepatitis E vaccine antigen P179

作　　者：迟祥[1] 李铮[1] 贾媛[1] 唐剑光[1] 宋昊[1] 陈子杨[1] 常东英[1] 迟春萍[1] 

机构地区：[1]长春生物制品研究所有限责任公司,吉林长春130062

出　　处：《中国生物制品学杂志》2016年第11期1126-1131,共6页Chinese Journal of Biologicals

摘　　要：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5.0×10^5-2.0×10^6CCU/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 perform characterization analysis on recombinant hepatitis E vaccine（HEV）antigen. Methods Three batches of bulk of r HEV antigen P179 with both HPLC and electrophoretic purities of 100% were determined for relative molecular mass and dimer content by SDS-PAGE, for amino acid component by OPA-FMOC automatic precolumn derivative amino acid-Amino Quant analysis, for amino acid sequence at C-terminus by electrospray ionization-quadrupole time-of-flight-mass spectrometry（ESI-Q-TOF-MS）method, and that at N-terminus by Edman degradation method, and for secondary structure by circular dichroism spectrometer. The protein particles were observed by transmission electron microscopy（TEM）with negative staining, while the antigen activity was determined by double antibody sandwich ELISA.Results The relative molecular masses of three batches of r HEV antigen P179 were 17 500 - 21 300, while dimer accounted for more than 85 % of total protein. The amino acid component, amino acid sequences at C- and N-terminuses, peptide map and isoelectric point were consistent with those expected. The α-helix accounted for 4. 8% -5. 6% of the secondary structure, while β-sheet for 34. 8% - 36. 4%, β-turn for 20. 8% - 22. 2%, and the irregular coiling for 37. 7% - 38. 0%. Uniform protein particles at sizes of about 20 nm were observed by TEM. The antigen activity ratios were 5. 0 × 10^5- 2. 0 × 10^6 CCU / mg. Conclusion The three batches of r HEV antigen P179 showed homogeneous structural characters, which were consistent with that designed. It laid a foundation of industrialization of the vaccine.

关 键 词：戊型肝炎疫苗 抗原 表征 

分 类 号：R373.21[医药卫生—病原生物学] R392.33[医药卫生—基础医学]