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High-titer culture method for phage and application

A culture method and phage technology, applied in the field of genetic engineering

Pending Publication Date: 2021-09-14
JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The lysis methods of most phages are only theoretical speculations based on gene comparison analysis, and there is no clear experimental confirmation

Method used

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  • High-titer culture method for phage and application
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  • High-titer culture method for phage and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Compensation host construction for expressing gene17.5 and gene19.5 encoded proteins

[0061] 1. Construction of recombinant plasmids

[0062] According to the T7 phage genome sequence, primers F4 and R4 were designed against the upstream and downstream sequences of gene17.5, and entrusted to GenScript to synthesize them. NcoI restriction sites were added to the 5' end of the upstream primer, and a His Tag tag was added to the 5' end of the downstream primer to stop a. Primers F5 and R5 were designed against the upstream and downstream sequences of gene19.5, a start codon was added to the 5' end of the upstream primer, and a stop codon, His Tag tag, and BamHI restriction site were added to the 5' end of the downstream primer. PCR amplification products were identified by agarose gel electrophoresis, gene17.5 with a size of about 200bp, gene19.5 with a size of 150bp, see figure 2 (A). Using the amplified gene fragment as a template, use primers F4 and R5 to...

Embodiment 2

[0065] Example 2 Gene17.5, gene19.5 double deletion T7 phage rescue

[0066] 1. Extraction of T7 phage genome

[0067] Centrifuge at 5000 rpm for 15 min to collect the T7 phage culture supernatant, add RNase A and DNase I at a final concentration of 1 μg / mL, react at 37 °C for 1 h, add 1 / 4 volume of PEG-NaCl solution (20% (w / v) PEG -8000, 2.5M NaCl), shake well and place at 4°C for 2-3h, centrifuge at 12000g for 30min to collect the precipitate. With 5mL SM solution (NaCl 5.8g, MgSO 4 .7H 2 O 2g, 1MTris-HCl (PH7.5) 50mL, 2% gelatin 5mL, dilute to 1L) to resuspend the pellet, add 50μL 10% SDS, 50μL 0.5MEDTA, digest at 65°C for 30min, add an equal volume of phenol / chloroform / iso Amyl alcohol (25:24:1), extracted 2 times. Transfer the supernatant and extract twice more with an equal volume of chloroform / isoamyl alcohol (24:1). Take the supernatant, add an equal volume of isopropanol to precipitate, centrifuge at 12000g, collect the precipitate, wash the precipitate with 1mL ...

Embodiment 3

[0074] Example 3 T7-△holin high titer culture technology

[0075] 1. Preparation of double gene deletion phage seed solution

[0076] Compensation host strains cryopreserved in glycerol were streaked on the LB solid medium plane and cultured overnight at 37°C. Pick a single colony from the plate, inoculate 5mL LB culture medium, culture overnight at 37°C and 200 rpm, take 3mL overnight culture and inoculate 300mL LB culture medium, cultivate to OD 600 =0.5 or so, inoculate positively identified T7-del17.5 / 19.5 phages, culture with shaking at 37°C and 150 rpm for 2 hours, add IPTG at a final concentration of 10mmol / L to induce the expression of gene17.5 and gene19.5 encoded proteins, Until the bacterial solution changed from turbid to clear, the double-layer agar method was used to measure the phage titer on a 100mmol / L IPTG plate, and the concentration of the cultured phage was adjusted to 1×10 11 pfu / mL, and add formaldehyde solution at a ratio of 4‰, and store at 4°C for l...

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Abstract

The invention provides a high-titer culture method for a T7 phage and application of the high-titer culture method. According to the high-titer culture method and the application thereof, recombinant Escherichia coli for expressing a gene 17.5 and gene 19.5 in-series coded protein is constructed by using a genetic engineering operation method and serves as a compensation host, then, a gene17.5 and gene19.5 double-gene deleted strain phage is rescued from the compensation host through a reverse genetic method, and the double-gene deleted T7 phage is copied and enriched in the host after infecting the compensation host and splits the host after the expression of the gene 17.5 and 19.5 coded protein is induced. The aim of high-titer culture is achieved by delaying the splitting time of the host and increasing the number of replication cycles of the phage in the host.

Description

technical field [0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to a method and application of high titer culture of bacteriophage, and more specifically relates to the construction of a compensating host, reverse genetic rescue of T7 phage with double gene deletion of gene17.5 and gene19.5, And on this basis, a high-titer culture method for phage was established. Background technique [0002] Phage is a virus that infects bacteria. Its host recognition is highly specific, its structure is simple, its number of genes is small, and it is easy to be manipulated by diverse groups. Therefore, it has become an important model system for molecular biology research. A phage particle can kill billions of bacteria, and this powerful bactericidal ability makes it a potential alternative to antibiotics and has attracted much attention. In 1985, G.P Smith founded the phage surface display technology, which can organically com...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N1/21C12R1/19C12R1/92
CPCC12N7/00C07K14/005C12N2795/10252C12N2795/10222
Inventor 徐海李玲李睿婷郭子杰洪伟鸣朱善元
Owner JIANGSU AGRI ANIMAL HUSBANDRY VOCATIONAL COLLEGE
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