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

发布时间：2018-09-18 12:14

【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 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。
[Abstract]:In order to develop a new type of hepatitis E genetic engineering vaccine, the recombinant hepatitis E virus-like particles were expressed in Hanson's yeast expression system, and the recombinant hepatitis E vaccine engineering strain HP/HEV 2.3 was successfully constructed. The fermentation conditions and purification process of the strain were studied. The recombinant Hansen yeast hepatitis E virus-like granules were prepared by cell breakage, clarification and ultrafiltration, silica gel adsorption and desorption, ultrafiltration, concentration and exchange, chromatographic purification and sterilization filtration. The purity of the recombinant Hansen yeast hepatitis E virus-like granules was 99% and the purity of the recombinant Hansen yeast hepatitis E virus-like granules was 33%. The theoretical size of hepatitis virus particles was 32 nm; the gene sequence was consistent with the theory; SDS-PAGE analysis showed that the molecular weight of the expressed foreign protein was 56 K Da, accounting for 26% of the total cell protein, and the expression level was 1.0 g/L fermentation broth; Western blotting and ELISA activity detection were performed. The results of ED_ (50) and mice immunization efficacy test showed that the recombinant yeast hepatitis E virus-like granule had good antigenicity and immunogenicity, and could be used to produce a new type of genetic engineering vaccine for hepatitis E.
【作者单位】：大连汉信生物制药有限公司;复旦大学生命科学学院;大连市血液中心;
【基金】：辽宁省企业技术中心专项资金(No.2011623)资助~~
【分类号】：R392

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