目的制备MDCK宿主细胞残留蛋白多克隆抗体,并进行纯化及初步应用,为进一步研制宿主细胞残留蛋白检测试剂盒奠定基础。方法无血清悬浮培养MDCK细胞,采用不同方法(反复冻融、裂解液、超声)裂解细胞获得全细胞蛋白抗原。将抗原与弗氏佐剂...目的制备MDCK宿主细胞残留蛋白多克隆抗体,并进行纯化及初步应用,为进一步研制宿主细胞残留蛋白检测试剂盒奠定基础。方法无血清悬浮培养MDCK细胞,采用不同方法(反复冻融、裂解液、超声)裂解细胞获得全细胞蛋白抗原。将抗原与弗氏佐剂乳化后经背部多点皮下免疫家兔,间接ELISA法检测血清抗体效价,当抗体效价≥1×106时,经家兔颈动脉采血,获得含多克隆抗体的血清。血清先经硫酸铵盐析粗提,再经DEAE-Sepharose Fast Flow和Protein A精纯,获得多克隆抗体,分析抗体蛋白回收率、纯度及抗原覆盖率。应用制备的多克隆抗体,Western blot法检测MDCK细胞培养流感病毒纯化工艺各阶段样品的宿主细胞残留蛋白。结果采用不同方法裂解细胞与不同离心力制备的抗原无区别,免疫家兔后,血清抗体效价最高可达320 000;纯化后的多克隆抗体纯度> 94%,DEAE-sepharose Fast Flow柱纯化效果优于Protein A柱;MDCK细胞培养流感病毒纯化初期存在较多的宿主细胞蛋白,经多步骤纯化后,单价原液中未见残留蛋白,仅含流感病毒蛋白。结论成功制备了MDCK宿主细胞残留蛋白多克隆抗体,可定性分析纯化各阶段样品的残留蛋白。展开更多
Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, follow...Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.展开更多
文摘目的制备MDCK宿主细胞残留蛋白多克隆抗体,并进行纯化及初步应用,为进一步研制宿主细胞残留蛋白检测试剂盒奠定基础。方法无血清悬浮培养MDCK细胞,采用不同方法(反复冻融、裂解液、超声)裂解细胞获得全细胞蛋白抗原。将抗原与弗氏佐剂乳化后经背部多点皮下免疫家兔,间接ELISA法检测血清抗体效价,当抗体效价≥1×106时,经家兔颈动脉采血,获得含多克隆抗体的血清。血清先经硫酸铵盐析粗提,再经DEAE-Sepharose Fast Flow和Protein A精纯,获得多克隆抗体,分析抗体蛋白回收率、纯度及抗原覆盖率。应用制备的多克隆抗体,Western blot法检测MDCK细胞培养流感病毒纯化工艺各阶段样品的宿主细胞残留蛋白。结果采用不同方法裂解细胞与不同离心力制备的抗原无区别,免疫家兔后,血清抗体效价最高可达320 000;纯化后的多克隆抗体纯度> 94%,DEAE-sepharose Fast Flow柱纯化效果优于Protein A柱;MDCK细胞培养流感病毒纯化初期存在较多的宿主细胞蛋白,经多步骤纯化后,单价原液中未见残留蛋白,仅含流感病毒蛋白。结论成功制备了MDCK宿主细胞残留蛋白多克隆抗体,可定性分析纯化各阶段样品的残留蛋白。
基金National Institutes of Health Grant (AI059570 and AI077767)
文摘Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.
