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Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof

A technology of Escherichia coli and bacteria preparations, which is applied in the field of preparation of bacteriostasis preparations, and can solve problems such as hindering the screening of bacteriostasis agents

Inactive Publication Date: 2012-10-03
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since different phages have different lytic enzyme sequences, even the same gene sequence will play different roles in different phages, and the expression of some genes in vitro will be restricted by many conditions, which hinders the screening of truly lytic enzymes. bacteriostasis agent

Method used

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  • Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof
  • Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof
  • Bacteria-cracking preparation for effectively cracking escherichia coli as well as cracking method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, induction of phage and preparation of phage DNA

[0026] Through the method of phage induction, induce the lysogenic strain of Escherichia coli, obtain lytic phage particles, purify the phage, and extract the DNA of the phage; the specific operation is as follows:

[0027] Escherichia coli O157Min27 strain was inoculated into 5 mL of LB liquid medium, shaken overnight at 37°C, then added to fresh LB liquid medium at a volume ratio of 1:4, then added mitomycin C to a final concentration of 1.0 μg / mL, and continued shaking After shaking for 14 hours, add 0.1% chloroform, shake again for 15 minutes, centrifuge at 4000 g for 10 minutes, filter the supernatant to sterilize, and the filtrate contains Stx2 phage. Add MC1061 to LB medium for doubling dilution 10 -2 、10 -4 、10 -6 , take 200μl phage dilution and mix with 200μl host bacteria, and add 1mol / L CaCl 2 To a final concentration of 0.1mol / L, after incubating at 37°C for 15 minutes, add the mixture to ...

Embodiment 2

[0029] Embodiment 2, design 2 pairs of primer primers, PCR amplifies target gene fragment

[0030] Design 2 pairs of primers P1 and P2, use the DNA obtained in Example 1 as a template, apply PCR to amplify the corresponding gene fragments, and determine that the amplified fragments of the genes are 534bp and 501bp; the specific operations are as follows:

[0031] According to the DNA sequence of Escherichia coli O157Min27 phage in GenBank (NC_010237), 2 pairs of primers were designed. The primer sequences are shown in Table 1 to amplify 2 gene segments respectively. At the 34-534 amino acids of the position, BamH I and Hind III restriction sites were respectively introduced at the 5' ends of the upstream and downstream primers. Using phage DNA as a template, the target gene fragment was amplified according to conventional methods. After the reaction, 5 μL of the product was taken for agarose gel electrophoresis. PCR products were recovered using a PCR product recovery kit. ...

Embodiment 3

[0034] Embodiment 3, the construction of recombinant plasmid

[0035] Using the prokaryotic expression system pET-28a(+), the DNA amplified in Example 2 was used to construct recombinant plasmids, and sequence analysis and comparison were performed to determine the recombinant plasmids pET-28a(+)-P1 and pET-28a( +) P2; the specific operation is as follows:

[0036] The PCR amplification product of Example 2 was connected to the pMD-18T vector (Takara Company) for sequencing. The designed primers include a BamH I restriction site upstream and a Hind III restriction site downstream. The PCR product digested with BamHI and Hind III was ligated with pET-28a (+), T4 DNA ligase (Takara Company) overnight at 10-16°C. The ligated product was transformed into Escherichia coli DH5α, cultivated overnight at 37°C, and the plasmid was extracted. The identified positive recombinant plasmids were sequenced by digestion with BamH I and Hind III, and the positive recombinant plasmids pET-28...

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Abstract

The invention discloses a bacteria-cracking preparation for effectively cracking escherichia coli as well as a cracking method and an application thereof. The bacteria-cracking preparation is prepared by the following steps of: designing a primer; using PCR (polymerase chain reaction) to amplify catenase gene in lysogenic escherichia coli for inducing lytic phage to obtain a target segment; connecting the target segment with pET-28a(+) carrier to obtain recombinant expression plasmid; introducing the recombinant expression plasmid to escherichia coli BL21(DE3) to express, thus obtaining positive transformant; expressing the recombinant fusion protein by induction; performing purification and induction to obtain purified recombinant protein, and screening the recombinant expression protein with cracking activity to obtain the bacteria-cracking preparation. The cracking method comprises the step of adopting 0.8% of beta-mercaptoethanol or 10mM of cysteine as reducing agent at pH of 8.0 and temperature of 37 DEG C, and 20mM of sodium acetate buffer liquid at pH of 5.2. The bacteria-cracking preparation obtained by the method provided by the invention has strong cracking specificity and high cracking efficiency for various serotype escherichia coli, and can effectively kill the escherichia coli.

Description

technical field [0001] The invention relates to a method for preparing a lysing preparation in the field of biotechnology, in particular to a lysing preparation for efficiently lysing Escherichia coli and its lysing method and application. Background technique [0002] Escherichia coli mainly parasitizes in the intestinal tract of humans and animals, and most of them are normal intestinal flora, but under certain conditions, they can become pathogenic bacteria, causing intestinal pathogenicity and extraintestinal pathogenicity, manifested as diarrhea and bleeding enteritis, septicemia, poultry air sac inflammation, synovitis and granuloma, etc. Among them, the typical serotype representative of intestinal pathogenicity is O157, which is a major serotype of Enterohemorrhagic Escherichia coli (EHEC), and is a foodborne zoonotic pathogen that can be transmitted through contaminated food. It can cause food poisoning in humans, and even cause death in serious cases. Therefore, i...

Claims

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Application Information

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IPC IPC(8): C12N15/70A61K38/16A61P31/04C12R1/19C12R1/92
Inventor 严亚贤孙建和杨曦
Owner SHANGHAI JIAO TONG UNIV
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