Hoatlin M E, Kew O M, Renz M E
J Virol. 1987 May;61(5):1442-7. doi: 10.1128/JVI.61.5.1442-1447.1987.
Poliovirus type 1 cDNA was prepared from viral RNA encoding the VP1 capsid region of the virus by using a specific DNA primer and was cloned in Escherichia coli. DNA fragments corresponding to VP1 amino acid positions 129 to 302 (pPM5k3), 52 to 302 (pPMhae3), and 24 to 129 (pPMDxba) were incorporated into plasmid vectors designed to express Trp LE-poliovirus VP1 fusion proteins under the control of the inducible tryptophan promoter-operator system. Induction of bacterial cultures containing the plasmids resulted in the production of fusion proteins which accounted for 21% (pPMhae3), 68% (pPM5k3), and 27% (pPMDxba) of the total cell protein. The proteins were purified, and each reacted with polyclonal antibodies raised against intact virions as measured by an enzyme-linked immunosorbent assay. The sera from rabbits immunized with the bacterially produced fusion proteins pPMDxba and pPMhae3 contained poliovirus-neutralizing antibodies.
通过使用特异性DNA引物,从编码病毒VP1衣壳区域的病毒RNA中制备1型脊髓灰质炎病毒cDNA,并将其克隆到大肠杆菌中。将对应于VP1氨基酸位置129至302(pPM5k3)、52至302(pPMhae3)和24至129(pPMDxba)的DNA片段整合到质粒载体中,这些质粒载体设计用于在可诱导的色氨酸启动子-操纵子系统的控制下表达Trp LE-脊髓灰质炎病毒VP1融合蛋白。含有这些质粒的细菌培养物经诱导后产生融合蛋白,其分别占总细胞蛋白的21%(pPMhae3)、68%(pPM5k3)和27%(pPMDxba)。对这些蛋白进行纯化,通过酶联免疫吸附测定法检测,每种蛋白都能与针对完整病毒粒子产生的多克隆抗体发生反应。用细菌产生的融合蛋白pPMDxba和pPMhae3免疫的兔血清中含有脊髓灰质炎病毒中和抗体。