PDF(Pubmed)
Abstract：
Bovine tuberculosis caused by Mycobacterium bovis, is a significant global pathogen causing economic loss in livestock and zoonotic TB in man. Several vaccine approaches are in development including reverse vaccinology which uses an unbiased approach to select open reading frames (ORF) of potential vaccine candidates, produce them as recombinant proteins and assesses their immunogenicity by direct immunization. To provide feasibility data for this approach we have cloned and expressed 123 ORFs from the M. bovis genome, using a mixture of E. coli and insect cell expression. We used a concatenated open reading frames design to reduce the number of clones required and single chain fusion proteins for protein pairs known to interact, such as the members of the PPE-PE family. Over 60% of clones showed soluble expression in one or the other host and most allowed rapid purification of the tagged bTB protein from the host cell background. The catalogue of recombinant proteins represents a resource that may be suitable for test immunisations in the development of an effective bTB vaccine.
摘要：
由牛分枝杆菌引起的牛结核病，是一种重要的全球病原体，会导致牲畜的经济损失和人类的人畜共患结核病。几种疫苗方法正在开发中，包括反向疫苗学，它使用无偏见的方法来选择潜在候选疫苗的开放阅读框（ORF）。将它们作为重组蛋白产生，并通过直接免疫评估其免疫原性。为了提供这种方法的可行性数据，我们从牛分枝杆菌基因组中克隆并表达了123个ORF，使用大肠杆菌和昆虫细胞表达的混合物。我们使用串联的开放阅读框设计来减少已知相互作用的蛋白质对所需的克隆和单链融合蛋白的数量，例如PPE-PE家族的成员。超过60%的克隆在一个或另一个宿主中显示出可溶性表达,并且最允许从宿主细胞背景中快速纯化标记的bTB蛋白。重组蛋白的目录代表了在开发有效的bTB疫苗中可能适用于测试免疫的资源。
