T7噬菌体尾丝蛋白随机进化文库的构建

为筛选遗传背景清晰、裂解性能优良、宿主识别谱广泛的噬菌体用于抗菌产品开发，满足畜禽减抗、无抗养殖需求，通过易错PCR技术定向改变T7噬菌体尾丝蛋白羧基末端基因序列，并构建T7噬菌体尾丝蛋白随机进化文库，为筛选识别不同宿主的T7噬菌体奠定基础。提取T7噬菌体基因组，用SfiⅠ进行单酶切，回收SfiⅠ位点左侧基因组。常规PCR扩增尾丝蛋白羧基末端基因序列(TF-ct,约350 bp)以及gene17至T7基因组右侧末端基因序列(约4 000 bp)。以回收的TF-ct片段为模板，进行易错PCR扩增，将随机突变的TF-ct基因片段连接T载体，构建质粒文库。从质粒文库中用SfiⅠ/SphⅠ双酶切出TF-ct片段，并与SfiⅠ位点左侧基因组片段、gp17右侧基因组片段进行连接，利用包装蛋白拯救出T7噬菌体尾丝蛋白随机进化文库。结果易错PCR成功扩增TF-ct基因片段，并连接T载体构建质粒文库；同时随机突变的基因正确插入T7噬菌体基因组相应位置，成功拯救出尾丝蛋白定向进化T7噬菌体文库。从噬菌体文库中随机挑选15个克隆进行序列分析，目的基因的突变率在1.23%～2.16%;尾丝蛋白loop区域的氨...

Construction of T7 phage tail fiber random evolution library

In order to satisfy the need of non-antibiotic livestock breeding, it is urgent to obtain new lytic bacteriophages with high-efficiency lysis, clear genetic background and broad host range, which can be used for anti-bacteria approaches. This study aimed to construct a random mutant library of the carboxy-terminal gene of T7 phage by using error-prone PCR method, so that the broad-host-range phages would be selected and identified from the library. The whole genome of T7 phage was extracted, and a 33 kb of left arm of genome was recovered after SfiⅠ digestion. The carboxy-terminal of T7 phage tail fiber (TF-ct, 350 bp) as well as the downstream arm (4 000 bp) from gene 17 were amplified by PCR method, respectively. TF-ct was used as templet for error-prone PCR amplification to obtain random mutant fragments, and the PCR products were connected with pMD19-T simple vector to construct a plasmid library. The SfiⅠ and SphⅠ enzyme site ready fragments were prepared by digesting plasmid library, and then connected with the 33 kb of genome left arm and 4 000 bp of gene 17 downstream arm to rescue T7 phage tail fiber random evolution library. Random mutant TF-ct gene fragments were successfully amplified by error-prone PCR, and both plasmid library and T7 phage library were correctly constructed. Fifteen phage clones were randomly picked from the library for DNA sequencing analysis, the results showed that the mutation rate of TF-ct gene was 1.23% to 2.16%. The amino acid mutant in loop region of tail fiber terminal led to the change of adsorption efficiency of T7 phage towards its host bacteria. The random evolution library of T7 phage tail fiber carboxy-terminal was successfully constructed, and further identified the relationship between the amino composition of tail fiber terminal loop region and T7 phage host recognition.