Abstract:

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.

Key words: T7 phage, tail fiber, random evolution, host recognition