福氏2a痢疾杆菌侵袭相关基因的克隆与表达

以福氏2a 痢疾杆菌大质粒为材料,粘粒(cosmid)为载体构建大质粒基因库。用探针菌落原位杂交,质粒分析及酶切、Southern blot、Western blot 等方法进行鉴定。获得了四株表达侵袭相关蛋白的克隆株,而且它们表达出与侵袭活性密切相关的接触性溶血活性。

THE CLONING AND EXPRESSION OF INVASIONASSOCIATED GENES OF SHIGELLA FLEXNERI SEROTYPE 2a

The pathogenicity of Shigella is correlated with their ability to penetrate and multiply within cells of the human colonic epithelium.A large plasmid is found in virulent isolates of Shigella sp which encodes functions essential for invasion of mammalian cell.In this report cosmid pIB_8 was used,as a vector,to clone the plasmid DNA fragments of Shigella flexneri 2a and shotgun cloned plasmid DNA directly into Ecoli DH1 and HB101 recipients. Recombinant clones were screened by in situ hybridization with 17 kb DNA probe derived from EcoR I fragment of Sh flexneri 5 large plasmid.To demonstrate that the recombinant clones produced peptides,Western blot analysis of whole bacteria extracts was performed. We succeeded in isolating four independent recombinants which could express invasion-asso- ciated protein antigens.The four recombinants expressed contact hemolytic activity.