大肠杆菌噬菌体P88尾丝蛋白受体识别关键氨基酸位点的确定

Identification of the key amino acid sites for receptor recognition of Escherichia coli phage P88

温和噬菌体P88由溶原性大肠杆菌K88诱导得来,前期研究结果表明其尾丝蛋白的716-746位氨基酸为其受体识别的关键区域。本研究旨在确定该关键区域内的关键氨基酸位点。采用大肠杆菌无痕缺失的方法以溶源大肠杆菌K88的基因组为操作对象,将P88尾丝蛋白中的第716、719、721、722、725、730、734、736、744和746位的氨基酸分别定点突变为丙氨酸,丝裂霉素C诱导定点突变溶原菌K88,之后用P88的宿主菌DE048筛选和纯化定点突变噬菌体,并测定定点突变噬菌体宿主谱。在构建的10株大肠杆菌K88定点突变株中,有5株可以诱导出与亲本噬菌体P88具有相同宿主谱的噬菌体突变株,然而尾丝蛋白第719、721、730、734和736位氨基酸定点突变的K88突变株无法用DE048筛选出突变噬菌体粒子,因此推测噬菌体尾丝蛋白第719、721、730、734和736位氨基酸为其受体识别的关键氨基酸位点。本研究为温和噬菌体基因和宿主谱的改造提供了理论基础。

The temperate phage P88 is induced from lysogenic Escherichia coli K88, and previous studies showed that the 716-746^th amino acids of its tail fiber protein were the key region for receptor recognition. This study was to identify the key amino acid sites within this key region. The 716^th, 719^th, 721^th, 722^th, 725^th, 730^th, 734^th, 736^th, 744^th and 746^th amino acid sites in the P88 tail fiber protein were replaced with alanine respectively by manipulating the E. coli K88 genome. The site-directed mutants of phage P88 were screened and purified using DE048 after induction of site-directed lysogen K88 mutants with mitomycin C, and the host range of site-directed P88 mutants were determined. Five among the ten mutants of E. coli K88 were able to induce P88 mutants with the same host range as that of the parental phage P88, but five K88 mutants at the 719^th, 721^th, 730^th, 734^th and 736^th amino acid sites of P88 tail fiber protein modification could not obtain phage particles using the same E. coli DE048. Therefore, it was speculated that the 719^th, 721^th, 730^th, 734^th and 736^th amino acids of P88 tail fiber protein were the key amino acid sites for receptor recognition. This study provides a theoretical basis for modification of gene and the host range of temperate phage.