噬菌体φ297切除酶(xis)基因的克隆和序列分析

Cloning and Sequence Analysis of Excisionase(xis) Gene of Phage φ297

目的:克隆噬菌体φ297 切除酶(xis)基因,并对其进行遗传与变异研究。方法:提取埃希氏大肠杆菌O157:H7 菌株EH297染色体DNA,采用步移PCR方法寻找目的基因,并通过克隆、亚克隆、DNA测序等分子生物学方法获得切除酶基因,通过序列分析软件对此基因进行分析。结果:克隆获得噬菌体φ297编码的切除酶基因(xis)的完整序列,它的长度是255 bp,编码了一个84个氨基酸组成的蛋白质(Xis),将它们的序列与λ噬菌体的切除酶家族的其它成员进行了比较。其结果是噬菌体φ297的切除酶基因(xis)与噬菌体VT1-Sakai的切除酶基因(xis)只有4个核苷酸的不同,而Xis蛋白与噬菌体VT1-Sakai的Xis蛋白是一样的,与噬菌体933W的Xis蛋白只有47.2%的相似性。结论:噬菌体φ297编码的切除酶基因(xis)与λ噬菌体的切除酶基因同源。

Objective:To clone and analyse of excisionase (xis) gene of phage φ297.Method:Total lysogenic bacteriophages genomic DNA of EHEC O157:H7 strains EH297 were extracted by centrifugation.Several approaches such as “Siebert PCR method for walking in uncloned DNA”,Clone and reclone of PCR products,DNA sequencing and data analysis were used in this study.Results:The complete sequence of the excisionase genes have been gotten that encoded by the phages φ297 in lysogenic bacteriophages.The xis gene is 255 bp long and codes for a protein of 84 amino acids.The nucleotide sequence of xis gene of phages φ297 are differing in only 4 nucleotides from nucleotides of phage VT1-Sakai.The Xis protein is identical with the Xis protein of the phage VT1-Sakai,which shows a similarity of 47.2% to the Xis of phage 933W.Conclusion: The xis gene of phage φ297 is similar to the other members of the lambdoid family.This study indicated that phage φ297 belong to lambdoid phages.