猫疱疹病毒1型病毒样颗粒在酵母中的制备及体外组装  

Preparation of feline herpesvirus type 1 VLPs in yeast and assembled in vitro

作　　者：赵梦杉 吕熙 王运航 裴辰辰 刘艺佳 郭慧琛 孙世琪[2] 杨丰利[1] 董虎[2] ZHAO Mengshan;LU Xi;WANG Yunhang;PEI Chenchen;LIU Yijia;GUO Huichen;SUN Shiqi;YANG Fengli;DONG Hu(College of Animal Science and Technology,Yangtze University,Jingzhou 434025,China;State Key Laboratory for Animal Disease Control and Prevention//College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730000,China;College of Life Sciences,Yulin University,Yulin 719000,China)

机构地区：[1]长江大学动物科学技术学院,湖北荆州434025 [2]中国农业科学院兰州兽医研究所兰州大学动物医学与生物安全学院动物疫病防控全国重点实验室,甘肃兰州730000 [3]榆林学院生命科学学院,陕西榆林719000

出　　处：《中国兽医科学》2024年第7期866-872,共7页Chinese Veterinary Science

基　　金：国家重点研发计划项目(2022YFD1800603,2021YFD1800300);国家自然科学基金项目(32072859,32072847,32002272)。

摘　　要：为研发猫疱疹病毒1型(FHV-1)病毒样颗粒(VLPs)疫苗,将FHV-1 g D基因和自组装蛋白I301基因序列融合后克隆到pPink-HC载体中,由毕赤酵母表达重组g D蛋白。随后观察不同pH对蛋白质表达和VLPs组装效果的影响。结果显示,经SDS-PAGE和Western-blot鉴定,发现培养基pH3.0条件下毕赤酵母表达出了完整的重组g D蛋白,而pH6.0条件下表达的蛋白分子质量小于预计大小;另外,组装液pH8.0时VLPs的组装效果优于pH6.0、7.0。结果表明,酸性环境可有效抑制毕赤酵母内蛋白酶对重组g D蛋白的水解作用,重组g D蛋白可在弱碱性环境中利用自组装蛋白I301在体外完成组装,为FHV-1 VLPs疫苗制备奠定了基础。To develop virus-like particles(VLPs)vaccines against feline herpesvirus type 1(FHV-1),FHV-1 g D gene was fused with self-assembling protein I301 gene and then cloned into p Pink-HC vector.The recombinant g D protein was expressed in Pichia pastoris.The effects of different p H on protein expression and VLPs assembly were then observed.The results showed that P.pastoris expressed intact recombinant g D protein at p H3.0,which was identified by SDS-PAGE and Western-blot,while the molecular weight of protein expressed under p H6.0 condition was smaller than expected.In addition,the assembly effect of VLPs at p H8.0 was better than that of p H6.0 and 7.0.These results showed that acidic environment could effectively inhibit the hydrolysis of recombinant g D protein by proteases in P.pastoris.Recombinant g D protein can be assembled into VLPs in vitro by using self-assembled protein I301 in a weak alkaline environment,which lays a foundation for preparation of FHV-1 VLPs vaccine.

关 键 词：猫疱疹病毒1型 病毒样颗粒 酵母表达系统 g D基因 

分 类 号：S852.659.1[农业科学—基础兽医学]