毕赤酵母分泌表达嵌合HEV表位的HBcAg颗粒

Secretion Expression of Hepatitis B Core Antigen Particles Harboring HEV Receptor-associated Epitope in Pichia pastoris

在毕赤酵母分泌表达嵌合有HEV受体相关表位12A10的HBcAg蛋白,经甲醇诱导后的培养液上清通过切向流浓缩、更换缓冲液后,进行疏水层析纯化.CsCl等密度梯度离心测得分泌的重组颗粒的密度为1.32 g/mL.透射电镜观察显示,纯化的重组颗粒为均一的直径30 nm左右的空心颗粒.小鼠免疫实验表明,纯化颗粒免疫8周后鼠血清中的特异性12A10抗体滴度可达到1.6×105,并且重组颗粒较好地呈递了HEV受体相关的非免疫优势表位.本文的结果为毕赤酵母胞外分泌表达其它大尺度的重组蛋白颗粒提供了参考,为研究携带表位多肽的疫苗载体提供了范例.

The hepatitis B virus core antigen harboring the HEV peptide was secretory-expressed in Pichia pastoris. The recombinant strains were cultivated and induced by methanol. After concentrating and exchanging buffer by crossflow filtration,the supernant was purified by hydrophobic interaction chromatography. Analysis of CsC1 iso-density gradient centrifuge indicated that the density of the recombinant protein was 1.32 g/mL. TEM analysis indicates that the HBc149-12A10 protein can self-assemble into particles,with approximately 30 nm in diameter. Mice were immunized with the recombinant protein,and 12A10-specific antibody was elicited more than the titer 1.6 × 10^5 after 8 weeks,moreover,the non-immunodominant epitope was highly presented in the recombinant particles. The results of our study may shed light of the secretion expression of large-size particles in P. pastoris and take insight into the rational design of the vaccine carrier.