人肠道病毒71型病毒样颗粒的制备及免疫原性评价  

Preparation and immunogenicity of virus-like particles of human enterovirus 71

作　　者：王文伟 胡海涛 蔡蓓蓓 仝光杰 何成 楼觉人 WANG Wen-wei;HU Hai-tao;CAI Bei-bei;TONG Guang-jie;HE Cheng;LOU Jue-ren(Shanghai Wellvax Co.,Ltd.,Shanghai 200051,China;不详)

机构地区：[1]上海至成生物科技有限公司,上海20005 [2]上海生物制品研究所有限责任公司,上海200051

出　　处：《中国生物制品学杂志》2021年第12期1406-1411,共6页Chinese Journal of Biologicals

基　　金：上海市科委生物医药领域科技支撑项目(13431900600)。

摘　　要：目的利用巴斯德毕赤酵母制备重组人肠道病毒71型(enterovirus 71,EV71)病毒样颗粒(virus-like particles,VLPs),并评价其免疫原性。方法将EV71的P1和3CD基因插入巴斯德毕赤酵母基因组中,构建EV71 VLPs表达质粒,转化巴斯德毕赤酵母KM71感受态细胞,Western blot法筛选高表达克隆,5 L发酵罐发酵,阳离子交换层析和凝胶体积排阻层析纯化EV71 VLPs。用不同剂量(0.05、0.5、5μg)的纯化EV71 VLPs免疫小鼠,ELISA法检测小鼠血清抗体滴度。结果EV71 VLPs表达质粒经双酶切鉴定证明构建正确。纯化的EV71 VLPs在电镜下可见大小均一、直径约为30 nm的典型VLPs;动态光散射分析显示,EV71 VLPs的流体力学直径为35.82 nm;经0.05、0.5和5μg EV71 VLPs免疫后,小鼠血清ELISA抗体的几何平均滴度(geometric mean titer,GMT)分别为119、800和3200,差异有统计学意义(P<0.01)。结论EV71 VLPs在巴斯德毕赤酵母中成功表达,且具有良好的免疫原性。Objective To prepare the virus-like particles(VLPs)of recombinant human enterovirus 71(EV71)by using Pichia pastoris and evaluate their immunogenicity.Methods The P1 and 3 CD genes of EV71 were inserted into the genome of P.pastoris,and the constructed expression vectors for EV71 VLPs were transformed to competent P.pastoris KM71 cells.The high expression clones were screened by Western blot and cultured in a 5 L fermenter,from which the EV71 VLPs were purified by cation exchange and size exclusion chromatography.Mice were immunized with the purified EV71 VLPs at dosages of 0.05,0.5 and 5μg respectively,of which the serum antibody titer was determined by ELISA.Results Restriction analysis showed that the expression vectors for EV71 VLPs were constructed correctly.Transmission electron microscopy(TEM)showed that EV71 VLPs were homogeneous spherical particles at a diameter of about 30 nm.Dynamic light scattering analysis showed that the hydrodynamic diameter of EV71 VLPs was 35.82 nm.The geometric mean titer(GMT)of ELISA antibodies in sera of mice immunized with 0.05,0.5 and 5μg EV71 VLPs were 119,800 and 3200 respectively.Conclusion EV71 VLPs were successfully expressed in P.pastoris,which showed good immunogenicity.

关 键 词：肠道病毒71型 手足口病 病毒样颗粒 毕赤酵母 免疫原性 

分 类 号：Q786[生物学—分子生物学]