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Construction method of recombinant bacteriophage

A construction method and phage technology, applied in the direction of phage, virus/phage, recombinant DNA technology, etc., can solve the problems of unclear gene function, unable to construct recombinant phage, etc., and achieve the effect of simple operation

Active Publication Date: 2021-07-20
GUANGXI UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] The purpose of the present invention is to provide a method for constructing recombinant phages to solve the problem that non-model filamentous phages cannot be constructed by existing methods due to lack of functional genome research and unclear gene functions

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  • Construction method of recombinant bacteriophage
  • Construction method of recombinant bacteriophage
  • Construction method of recombinant bacteriophage

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Embodiment

[0029] A method for constructing a recombinant phage, comprising the steps of:

[0030] (1) Extract the replicating DNA of filamentous phage, the filamentous phage RSCq of Ralstia solanacearum, the filamentous phage has not been reported and studied yet, its genome is single-stranded DNA, replicating double-stranded circular DNA A total of 7480bp, the sequence is shown in SEQ ID No.1;

[0031] (2) Preparation of Tn5 transposon DNA with the target gene: the target gene is the esterase elp104 DNA, which is controlled by the lac promoter and degrades the quorum sensing signal molecule of R. solanacearum, synthesized through commercial gene synthesis services, sequence See SEQ ID No.2;

[0032] The structural diagram of transposon DNA is shown in figure 1 The Tn5 transposon DNA is flanked by the recognition sequence of Tn5 transposase, and contains the replication initiation site ori required for the replication of filamentous phage replication DNA in Escherichia coli, and is us...

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Abstract

The invention relates to the technical field of molecular biology, in particular to a construction method of a recombinant bacteriophage. The construction method of the recombinant bacteriophage comprises the following steps: (1) extracting replicative DNA of the filamentous bacteriophage; (2) preparing a Tn5 transposon DNA with a target gene; (3) inserting the transposon DNA in vitro; (4) transforming escherichia coli DH5[alpha][lambda]pir; (5) preparing a mixed plasmid library; (6) transforming host cells; (7) screening recombinant bacteriophages; and (8) detecting the expression of the target gene. The construction method provided by the invention is simple to operate, can construct the recombinant filamentous phage without depending on the research on the functional genome of the target filamentous phage, and has important significance on the research on the application of the target recombinant filamentous phage.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for constructing a recombinant phage. Background technique [0002] Phages are viruses that specifically infect microorganisms such as bacteria, archaea, and algae. Recombinant phages can be obtained by genetically modifying phages; certain characteristics of bacteriophages can be strengthened, changed, or endowed through genetic modification, which has important application value in anti-bacterial infection, pathogen detection, and biological control of plant bacterial diseases. For example, the replacement of the T7-like phage tail gene changes its host range; another example is the insertion of a fluorescent protein coding gene or a luciferase gene in the phage genome, and the obtained recombinant phage can be used for rapid detection of pathogenic bacteria. At present, the genetic modification methods of tailed phages include homologous recombination in ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/63
CPCC12N7/00C12N15/63C12N2795/14121Y02A50/30
Inventor 郑德洪彭仕文袁高庆
Owner GUANGXI UNIV
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