Kuge S, Chisaka O, Kohara M, Imura N, Matsubara K, Nomoto A
J Biochem. 1984 Aug;96(2):305-12. doi: 10.1093/oxfordjournals.jbchem.a134839.
DNA sequences coding for the immunogenic capsid protein VP1 and/or VP3 of poliovirus strain LSc-2ab (Sabin 1) were prepared by digesting the cloned complementary DNA with restriction endonuclease PstI. The DNA fragments were inserted into the unique PstI site of Escherichia coli plasmid vectors pBR322, pKT 280 and/or pKT 287 that lay in the region expressed under control of the penicillinase promoter system. In the expression vectors, poliovirus sequences were designed to be read in phase and therefore to be expressed as fusion proteins with the bacterial peptides. In addition, the Escherichia coli tryptophan operon promoter-operator system was inserted upstream of the penicillinase system to obtain stronger expression of the poliovirus sequences. Escherichia coli transformed with these plasmids appeared to produce the antigenic polypeptides, which were detected by immunoprecipitation with antibodies to capsid proteins VP1 and/or VP3 followed by SDS-polyacrylamide gel electrophoresis.
通过用限制性内切酶PstI消化克隆的互补DNA,制备编码脊髓灰质炎病毒株LSc - 2ab(萨宾1型)免疫原性衣壳蛋白VP1和/或VP3的DNA序列。将DNA片段插入大肠杆菌质粒载体pBR322、pKT 280和/或pKT 287的唯一PstI位点,这些位点位于青霉素酶启动子系统控制下表达的区域。在表达载体中,脊髓灰质炎病毒序列设计为相位可读,因此可作为与细菌肽的融合蛋白表达。此外,将大肠杆菌色氨酸操纵子启动子 - 操纵子系统插入青霉素酶系统上游,以获得脊髓灰质炎病毒序列更强的表达。用这些质粒转化的大肠杆菌似乎产生了抗原性多肽,通过用针对衣壳蛋白VP1和/或VP3的抗体进行免疫沉淀,然后进行SDS - 聚丙烯酰胺凝胶电泳来检测。