禽网状内皮组织增生症病毒gp90蛋白在悬浮培养细胞中的表达及免疫原性分析
文献类型: 中文期刊
关键词: 禽网状内皮组织增生症病毒;;gp90蛋白;;真核表达;;293F细胞;;悬浮培养
期刊名称:中国家禽
ISSN: 1004-6364
年卷期: 2025 年
页码:
收录情况: 北大核心(2023版) ; ; 科技核心(2024版) ; ; 农林核心(2024版)
摘要: 为高效制备禽网状内皮组织增生症病毒(Reticuloendotheliosis virus,REV)囊膜蛋白gp90,试验利用PCR技术扩增获得REV流行毒株gp90基因,利用同源重组技术将该基因克隆入真核表达载体 p19-2 构建重组真核表达质粒 p19-2-6his-peptide-gp90-12his;在转染至293T细胞中进行表达特性初步验证后,将重组质粒转染至悬浮培养的293F细胞中进行表达制备,采用镍柱亲和层析技术纯化分泌到细胞上清中的gp90蛋白,对其进行Western blot检测;纯化的 gp90蛋白免疫 SPF鸡,通过检测血清抗体的情况鉴定 gp90蛋白的免疫原性。结果显示:利用 293F细胞悬浮培养系统获得了 REV 流行毒株的囊膜蛋白 gp90的高效分泌表达,经镍柱亲和层析技术纯化获得的重组蛋白浓度可达 731.91 μg/ml,其可被 REV gp90单克隆抗体特异性识别,具有良好的反应原性;该蛋白具有良好的免疫原性,其免疫鸡后可刺激产生特异性抗体,一免后血清抗体全部转阳,二免后血清抗体的平均ELISA效价可达7 067。研究表明,成功利用细胞悬浮培养系统制备了具有良好反应原性和免疫原性的REV gp90蛋白,为进一步开展REV候选疫苗及配套检测方法的研究奠定了基础。
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