Method for producing bacteriophage modified by random sequence insertion into bacteriophage screening protein

摘要

Preparing a bank for genetically modified bacteriophages, comprises transforming a host bacterium by using at least a DNA construction allowing the insertion of an oligonucleotide of which the sequence is produced randomly, in at least a gene encoding a targeting protein of a bacteriophage; infecting the transformed bacteria with the bacteriophage; placing the infected bacteria under conditions i.e. bacteriophages replicate, and that at least a part of oligonucleotides produced randomly; and collecting the recombinant bacteriophages which are replicated in the infected host bacteria. Preparing a bank for genetically modified bacteriophages, comprises transforming a host bacterium by using at least a construction of DNA allowing the insertion of an oligonucleotide of which the sequence is produced randomly, in at least a gene encoding a targeting protein of a bacteriophage; infecting the transformed bacteria with the bacteriophage; placing the infected bacteria under conditions i.e. bacteriophages replicate, and that at least a part of oligonucleotides produced randomly, able to be inserted in at least a gene encoding a targeting protein of the bacteriophage; and collecting the recombinant bacteriophages which are replicated in the infected host bacteria. Independent claims are included for: (1) a bank for recombinant bacteriophages able to be obtained by the process; (2) a process for introducing randomly produced oligonucleotide sequences in the sequence of the gene encoding a targeting protein of the bacteriophage, by retaining the identity of several internal segments D n to D n ? + 1 of the gene sequence, comprises conducting a PCR-error over the entire sequence using primers corresponding to the segments (F1) and (F2), by which the sequence is transferred at random along its entire length; selecting the obtained amplification product; conducting a high-fidelity PCR from the amplification products, by using pairs of primers corresponding to at least F1 and D n ? + 1, and F2 and D n for amplifying at least the regions F1-D n ? + 1 and D n-F2 of the mutated sequence of which the internal segments D n ? + 1 and D n are maintained their identity; conducting a high-fidelity PCR from the obtained amplification products using primers corresponding to F1 and F2; and purifying the obtained PCR products of which the size corresponds to the nucleotide sequence; (3) a construction of DNA to insert the randomly produced nucleotide sequence in the gene encoding the targeting protein of the bacteriophage by homologous recombination, comprising the amplification product, which is obtained by the process; (4) a bank of host bacteria of transformed bacteria using a number of constructions of DNA; and (5) a method for selecting a genetically modified bacteriophage allowing for targeting a cell of which the identity is known or unknown, comprising contacting the obtained genetically modified bacteriophages using the process with a cell whose identity is known or unknown, and selecting at least a recombinant bacteriophage contacting with the cell whose identity is known or unknown, able to infect the cell. ACTIVITY : Antibacterial. MECHANISM OF ACTION : None given. No biological data given.