致肠出血性EH297整合酶基因(int)的克隆与分析

Cloning and analysis of integrase gene (int) of EH297 as predominant cause of bloody diarrhea

目的 克隆并分析致肠出血性埃希氏大肠杆菌O15 7:H7菌株EH2 97的整合酶基因。方法 采用加接头的步移PCR方法 ,从溶源性噬菌体 φ2 97的染色体DNA上类似于噬菌体 933W的整合酶基因的一个 4 0个核苷酸开始的 ,进行了克隆、亚克隆、测序和序列分析等分子生物学研究。结果 得到了噬菌体 (2 97编码的整合酶基因(int)的完整序列 ,它的长度是 12 87bp ,编码了 4 2 8个氨基酸的Int蛋白质。将它们的序列与λ噬菌体的整合酶家族的其它成员进行了比较。发现噬菌体 φ2 97的整合酶基因 (int)与噬菌体VT1 Sakai的整合酶基因有 79%的相似性 ,噬菌体 φ2 97的Int蛋白与噬菌体VT1 Sakai的Int蛋白在氨基酸序列上有 82 %的相似性。N 末端的氨基酸区域是完全保守的 ,而中心区和C 末端则显示出高度的变异。结论 噬菌体 φ2 97属于λ噬菌体或它们的int基因来源于同一基因池。

Objective: To clone and analyse the integrase (int) gene of bacteriophages EH297. Methods: Molecular techniques were used such as Siebert PCR for walking from the 40 nucleotide of prophage 297, which was similar to that int gene of phage 933W to an unknown region in genomic DNA. A special adaptor was ligated to the ends of DNA fragments generated by digestion of genomic DNA with restriction enzymes that generates blunt ended fragments. Clone and reclone of PCR products, DNA sequencing and data analysis were used in this study. Results: The complete sequence of the intergrase gene (int) was obtained that encoded by the phages φ297. The int gene was 1287 bp long and codes for a protein of 428 amino acids. The nucleotide sequence of the int gene of phage φ297 was compared, showing the highest identity (79%) with the int gene of phage VT1-Sakai.The Int protein of the phages φ297 and VT1-Sakai were similar but not identical. The overall identity at the amino acid level was 82%.Aamino acid regions of N-terminal were completely conservative, whereas a much higher divergence was in the central and C-terminal region. Conclusion: The phage φ297 and VT1-Sakai have a common ancestor or that these genes are present in a common gene pool.