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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 k Da,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ED_(50)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。
In order to develop a novel genetically engineered hepatitis E vaccine, the recombinant Hepatitis E virus-like particle was expressed by the Hansenula expression system and the recombinant Hepatitis E vaccine engineering strain HP / HEV2.3 was successfully constructed. The fermentation conditions and purification process Were studied. The working seed batch is firstly fermented and cultured, and the cell culture after fermentation is collected; the cell culture is successively subjected to cell disintegration, clarification and ultrafiltration, adsorption and desorption of silica gel, concentration and exchange of ultrafiltration, chromatographic purification and sterilization filtration to obtain a recombinant Hansenula HEV-like particles, the purification yield was 33%, the purity of 99%; electron microscopy showed that the recombinant Hansenula HEV-like particles and the natural hepatitis E virus particle size of the theory, for 32 nm ; The sequence of the gene was consistent with the theory; SDS-PAGE analysis showed that the molecular weight of the expressed foreign protein was 56 kDa, which was 26% of the total cellular protein and the expression level was 1.0 g / L fermentation broth; Western blotting, ELISA activity test and mouse immunization efficacy test ED_ (50) results show that this recombinant Hansenula hepatitis E virus-like particles with good antigenicity and immunogenicity, can be used for the manufacture of E Hepatitis A New Genetic Engineering Vaccine.