口蹄疫病毒样颗粒在毕赤酵母中的表达与鉴定

Expression and characterization of foot-and-mouth disease virus-like particles in Pichia pastoris

摘要

[目的]口蹄疫病毒(FMDV)灭活苗在其生产过程中依然存在病毒泄漏风险,本文旨在研发具有与病毒粒子结构相似,且不含核酸的病毒样颗粒(virus-like particles,VLP),并通过毕赤酵母真核表达系统进行表达生产成本低廉的理想FMDVVLP疫苗。[方法]首先将FMDV的VP0、VP3和VP1表达盒插入到同一个pPink-HC质粒上,再电转化毕赤酵母,通过蔗糖密度梯度纯化获得制备FMDVVLP,并采用ELISA对其进行了定量。[结果]重组菌经过甲醇诱导,蛋白免疫印记(Western blot,WB)检测目的蛋白的表达;重组菌破碎后离心的上清液经过蔗糖梯度离心分离得到纯化的VLP;经动态光散射法(dynamic light scattering,DLS)和透射电子显微镜(transmission electron microscope,TEM)检测,可观察到直径为30nm的球形颗粒;经ELISA检测,VLP呈现出良好反应性,这表明所制备的VLP具备良好抗原性。[结论]采用单独表达VP0、VP3和VP1的方式在毕赤酵母中成功制备FMDVVLP,这为FMD疫苗的制备及其他小RNA病毒样颗粒的制备提供了试验依据。

Abstract

[Objectives] Inactivated vaccines against Foot-and-mouth disease virus (FMDV) still bear a risk of virus leakage during the virus propagation and production process. This paper aimed to develop virus-like particles (VLP) of FMDV, which have a structure similar to that of virus particles but lack nucleic acids. These VLP can be expressed through the Pichia pastoris eukaryotic expression system to produce an ideal and more cost-effective FMDV LVP vaccine. [Methods] The expression cassettes of VP0, VP3 and VP1 gene of FMDV were inserted into the same pPink-HC plasmid, and then FMDV VLP were produced by electroporating P. pastoris. FMDV VLPs was purified by sucrose density gradient ultracentrifugation, and quantified by ELISA. [Results] After induction with methanol, the expression of the target proteins could be detected by western blot (WB). Purified VLP could be isolated from the supernatant after cell disruption and sucrose gradient centrifugation. Spherical particles with a diameter of 30 nm were detected by dynamic light scattering (DLS) and transmission electron microscopy (TEM), and the antigenicity of the prepared VLP was determined by ELISA and showed a good response, which fully demonstrated that the prepared VLP possesses antigenicity. [Conclusions] FMDV VLP were successfully produced in P. pastoris by separately expressing VP0, VP3 and VP1, providing an experimental basis for the development of FMD vaccines and the production of other picornavirus virus-like particles.

关键词

口蹄疫病毒 / 病毒样颗粒 / 真核表达 / 毕赤酵母 / 疫苗

Key words

foot-and-mouth disease virus / virus-like particles / eukaryotic expression / Pichia pastoris / vaccine

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基金资助

甘肃省科技计划东西部科技协作专项(24CXNA030)

宁夏回族自治区重点研发计划(2024BBF02017)

2023兰州市人才创业创新项目(2023-RC-3)

江苏省农业科技自主创新资金(CX(22)2016)