戊型肝炎病毒样颗粒在重组汉逊酵母中的发酵表达、纯化及其免疫原性分析

Fermentation, purification and immunogenicity evaluation of hepatitis E virus-like particles expressed in Hansenula polymorpha

摘要:

为了研制戊型肝炎新型基因工程疫苗，利用汉逊酵母表达系统表达重组戊型肝炎病毒样颗粒，成功构建了重组戊型肝炎疫苗工程菌株HP/HEV2.3，对该菌株的发酵条件和纯化工艺进行了研究。先将工作种子批进行发酵培养，收集发酵后的细胞培养物；对其先后进行细胞破碎、澄清和超滤、硅胶吸附和解吸附、超滤浓缩换液、色谱纯化及除菌过滤，制得重组汉逊酵母戊型肝炎病毒样颗粒，纯化收率为33%，纯度达99%；电镜观察显示该重组汉逊酵母戊型肝炎病毒样颗粒与天然戊型肝炎病毒颗粒理论大小一致，为32 nm；基因序列与理论一致；SDS-PAGE分析结果表明其表达的外源蛋白质分子量与预期的目的蛋白质分子量大小一致，均为56 kDa，表达量占细胞总蛋白的26%，表达水平为1.0 g/L发酵液；Western blotting、ELISA活性检测及小鼠免疫接种效力试验ED50结果表明，此重组汉逊酵母戊型肝炎病毒样颗粒具有良好的抗原性和免疫原性，可用于制造戊型肝炎新型基因工程疫苗。

Abstract:

To develop a new recombinant hepatitis E vaccine, we used Hansenula polymorpha expression system to express recombinant hepatitis E virus-like particles (HEV VLPs), to construct a recombinant engineered strain HP/HEV2.3. The fermentation conditions and purification process were studied next. The first working seed lots were fermented in liquid culture, and the fermentation products were collected, then crushed, clarified, purified by ultrafiltration, silica gel adsorbed and desorbed, concentrated by ultrafiltration, purified by liquid chromatography and sterilized by filtration. The purity reached 99% with a yield of 33%. Electron microscopy analysis revealed that both the purified recombinant HEV VLPs from HP/HEV2.3 and natural hepatitis E virus particles appear identical of being 32 nm. The resulting DNA sequence obtained from VLPs is identical to the published HEV sequence. The SDS-PAGE analysis has revealed that the protein molecular weight of the HEV VLPs is 56 kDa, and the expression product HEV VLPs were accumulated up to 26% of total cellular protein. The expression level is 1.0 g/L. Western blotting, enzyme-linked immunosorbent assay (ELISA) results of the protein and ED50 of the vaccine showed that the HEV VLPs have good antigenicity and immunogenicity. In summary, the recombinant HEV VLPs from Hansenula polymorpha can be used in the manufacture of a new genetically engineered vaccine against hepatitis E.