[Expression and characterization of equid herpesvirus 1 glycoprotein D in Pichia pastoris]

ABSTRACT Equine herpesvirus 1 (EHV-1) has a worldwide distribution and causes serious damage to horse breeding. It is an agent of respiratory, reproductive and neurological disease outbreaks in young and adult equids. Viral envelope glycoprotein D (gD) is essential for binding and penetration into permissive cells and targeting the host immune system, inducing humoral and cellular immune responses, and is an appropriate antigen for use in vaccines and immunodiagnostics. The objective of this work was to express in Pichia pastoris and to characterize EHV-1 gD for later use as an antigen in immunodiagnostic techniques and formulation of recombinant vaccines. A DNA sequence encoding a truncated form of gDEHV-1 has been cloned into the P. pastoris expression vector pPICZαA. A protein of ~41 kDa was obtained as expected. The protein presented glycosylation between 4 kDa and 16 kDa, demonstrated by enzymatic deglycosylation. The recombinant protein was antigenically and immunogenically characterized by Western blot using equine polyclonal anti-EHV-1 antibodies, and by indirect ELISA in a murine model, demonstrating that the recombinant gD maintained epitopes similar to those of the native protein. These results suggest that gDEHV-1 is a promising antigen for use as an immunobiological in the control of EHV-1.

摘要：马疱疹病毒1型（Equine herpesvirus 1, EHV-1）呈全球分布，对马匹繁育造成严重危害，可引发马科动物幼体与成体出现呼吸、生殖及神经系统疾病暴发。该病毒的包膜糖蛋白D（gD）是介导病毒结合并侵入易感细胞、靶向调控宿主免疫系统、诱导体液与细胞免疫应答的关键蛋白，亦是疫苗研发与免疫诊断试剂开发的理想抗原。本研究旨在毕赤酵母（Pichia pastoris）中表达EHV-1 gD并对其进行特性鉴定，以期将其作为抗原应用于免疫诊断技术开发与重组疫苗研制。研究人员将编码截短型EHV-1 gD的DNA序列克隆至毕赤酵母表达载体pPICZαA中，成功获得了预期分子量约41 kDa的目标蛋白。经酶促去糖基化实验验证，该蛋白存在4 kDa至16 kDa区间的糖基化修饰。通过使用马源多克隆抗EHV-1抗体进行蛋白质免疫印迹（Western blot）分析，以及在小鼠模型中开展间接酶联免疫吸附试验（indirect ELISA），对重组蛋白的抗原性与免疫原性进行了表征，结果显示重组gD保留了与天然蛋白相似的抗原表位。上述研究结果表明，截短型EHV-1 gD可作为极具应用前景的免疫生物制剂，用于EHV-1的防控工作。