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作者从野型2a型福氏志贺菌2457T株的DNA中提取gua B的5’末端和gua A的3’末端,通过聚合酶链反应(PCR)将这两个区段扩增融合,构成△gua B-A等位基因.用外引物把克隆△gua B-A等位基因的唯一限制性位点导入温度敏感的pFM307A质粒,构成pFM726A载体质粒.用pFM726A通过同源重组把缺失突变导入2457T株,产生单突变△guaB-A CVD1204株.用p△virG在毒力基因virG上进行第二个缺失突变,构成双突变△gua B-A△virG CVD1205株.作者比较了CVD 1204株和另一2a型单突变株△aro A CVD1201(aro缺陷型)的侵袭力、细胞内生长及安全性,并就CVD1205株在豚鼠中的安全性、免疫原性及保护率作了评估.
The authors extracted the 5 ’end of gua B and the 3’ end of gua A from the DNA of wild type 2a Shigella flexneri 2457T strain, and amplified and fused these two segments by polymerase chain reaction △ gua BA allele. The only restriction site for the cloned △ gua BA allele was introduced into the temperature-sensitive pFM307A plasmid using an external primer to construct the pFM726A vector plasmid. The deletion mutation was introduced into the 2457T strain by homologous recombination with pFM726A Single mutant △ guaB-A CVD1204 strains with p △ virG in the virg gene virG a second deletion mutation to form a double mutant △ gua BA △ virG CVD1205 strains compared CVD 1204 strains and another 2a single mutation The invasiveness, intracellular growth and safety of strain aro A CVD1201 (aro defective) were evaluated and the safety, immunogenicity and protection of CVD1205 strain in guinea pigs were evaluated.