霍乱弧菌分型噬菌体VP3尾丝蛋白的表达及功能分析

目的研究霍乱弧菌分型噬菌体VP3的尾丝蛋白的受体结合功能,探讨VP3感染机制。方法分析VP3的尾丝蛋白gp44序列并克隆表达和纯化,以荧光显微镜和激光共聚焦显微镜观察尾丝蛋白与VP3敏感株和抗性株的结合能力。利用重叠PCR扩增、测定并分析对VP3有天然抗性的霍乱弧菌野生株的wav基因簇的序列。结果VP3的gp44与T7噬菌体尾丝蛋白gp17的同源性高。表达的重组蛋白His-gp44与大肠杆菌不能结合,与VP3敏感株和天然抗性株结合,敏感株的核心寡糖基因簇(wav)基因缺失后不能结合。能与His-gp44结合的敏感株和抗性株,其wav基因簇序列只有一个碱基的差异。结论VP3的gp44是尾丝蛋白,能与霍乱弧菌表面受体特异性结合。

Expression and functional analysis of the tail fiber protein of Vibrio cholerae typing phage VP3

To investigate the function for receptor-binding of the tail fiber protein gp44 of Vibrio cholerae typing phage VP3 and to test whether gp44 is the site of interaction with its receptor on V.cholerae cell surface,gp44 was cloned and expressed with 6-His tag added to its N-terminus. The purified His-gp44 in solution was then incubated with the strain susceptible or resistant to the lytic action of VP3, its whether it wuld bind the susceptible or resistant strains was observed by fluorescent microscopy and laser scanning co-focal microscopy. Meanwhile,the sequence of the wav gene cluster wav the wild type V.cholerae with natural resistant to VP3 was DCR,amplified,and sequcnced.It was found that His-gp44 located on the surface of both susceptible and resistant stains,but not on the surface of Escherichiawti. When one of the genes in gene cluster wav was deleted,the susceptible mutant showed no His-gp44 around its cell wall. The sequence of the gene cluster wav showed only one single base difference in susceptible and resistant strains to VP3. It is concluded from the above mentioned observations that the tail fiber protein gp44 of V. cholerae typing phage VP3 can bind specifically with the surface receptors on V. cholerae.