猪链球菌9型噬菌体裂解酶Ply5218最小功能域及关键氨基酸位点的鉴定

Identification of the minimum domains and the key amino acids of the lysin Ply5218 encoded by prophage from Streptococcus suis serotype 9

为了鉴定猪链球菌9型噬菌体裂解酶Ply5218的最小活性功能域及关键氨基酸位点,利用PCR技术对全长裂解酶Ply5218进行截短并对可能与活性相关的关键氨基酸位点进行定点突变,研究截短与突变后各个蛋白的活性并与全长蛋白活性对比。结果显示,截短片段Ply5218_(1-147)可在平板上形成裂菌圈,仍保持裂菌活性,而比该片段更短的截短片段则失去裂菌活性,推测Ply5218_(1-147)为Ply5218的最小活性相关功能域;第8、58、136和142位点的氨基酸突变后,裂菌活性部分降低,第34和144位点的氨基酸突变后,则彻底失去裂菌活性。研究结果为深入揭示Ply5218的裂菌机制和进一步改造裂解酶提供了基础数据。

PCR based deletion and point mutation were carried out to identify the minimal functional domain and the key amino acid sites of Streptococcus suis type 9 phage encoded lysin Ply5218.The target proteins were expressed in Escherichia coli and tested by plate lysis experiments.By comparing with the activity of the full-length lysin,the minimum functional domain and the key amino acids of Ply5218 were determined.The results showed that Ply5218 1-147 exhibited a strong lytic activity against Streptococcus suis HA9801,while the shorter fragments would lose the activity to lyses bacteria,which confirmed that fragment Ply5218 1-147 is essential for active Ply5218.Moreover,proteins changed at amino acid sites 8,58,136 and 142 had low bacteriolysis activities and those at 34 and 144 lost their lytic activities completely.