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Enzymatic antibiotic for killing gram-positive bacteria and preparation and use of enzymatic antibiotic

A sequence and protein technology, applied in the field of microbial chemistry, can solve the problems of low enzyme activity and difficulty in mass production

Inactive Publication Date: 2014-10-01
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are very few practical hydrolytic enzymes discovered so far, and most of these enzymes have low activity or are difficult to produce in large quantities, especially enzyme antibiotics that can specifically cleave multi-drug resistant Streptococcus and Staphylococcus aureus at the same time Not yet reported, the outstanding advantage of the present invention is exactly to have solved these several problems above

Method used

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  • Enzymatic antibiotic for killing gram-positive bacteria and preparation and use of enzymatic antibiotic
  • Enzymatic antibiotic for killing gram-positive bacteria and preparation and use of enzymatic antibiotic
  • Enzymatic antibiotic for killing gram-positive bacteria and preparation and use of enzymatic antibiotic

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1, design primer, PCR amplification target gene fragment

[0051] Refer to the gene sequence of Streptococcus suis strains in GenBank, design primers, use Streptococcus suis genomic DNA as a template, and use PCR technology to amplify the target gene. The size of the target gene is 738bp. The specific operation is as follows:

[0052] Primers were designed, the primer sequences are shown in Table 1, the target gene was amplified, and EcoR I and Hind III restriction sites were inserted at the 5' ends of the upstream and downstream primers, respectively. Using the genomic DNA of Streptococcus suis strain as a template, the target gene fragment was amplified according to conventional methods. After the PCR reaction, 10 μL of the product was taken for 1% agarose gel electrophoresis to check the size of the target band. The PCR product was recovered using a gel recovery kit.

[0053] Table 1 PCR primer sequence

[0054]

Embodiment 2

[0055] Embodiment 2, construction of recombinant plasmid pET-28a(+)-LySS7

[0056] Using the prokaryotic expression system pET-28a(+), construct a recombinant plasmid with the target gene obtained from the gel recovery kit in Example 1, perform sequence analysis and comparison, and determine the recombinant plasmid pET-28a(+)-LySS7 with the correct sequence; The operation is as follows:

[0057] The gel recovery product of Example 1 was connected to the vector pET-28a(+) for sequencing. The upstream and downstream of the designed primers respectively contain EcoR I restriction endonuclease cutting site and Hind III restriction endonuclease cutting site. The DNA and pET-28a(+) product digested by EcoR I and Hind III, at T 4 Under the action of DNA quick ligase (Fermentas company), the ligation was performed at 22° C. for 30 min. The ligated product was transferred into the cloning vector Escherichia coli DH5α, cultivated overnight at 37°C, and the plasmid was extracted. E...

Embodiment 3

[0058] Embodiment 3, prokaryotic expression of recombinant plasmid BL21 / pET-28a(+)-LySS7

[0059] The recombinant plasmid pET-28a(+)-LySS7 in Example 2 was transformed into the expression vector Escherichia coli BL21(DE3), and the positive clone BL21 / pET-28a(+)-LySS7 was screened for inducible expression and identification of the protein to obtain recombinant Protein, the molecular weight of the recombinant protein is determined to be 29.8kDa; the specific operation is as follows:

[0060] The positive recombinant plasmid pET-28a(+)-LySS7 was transformed into the expression vector Escherichia coli BL21(DE3). The positive clone BL21 / pET-28a(+)-LySS7 was obtained, and a single colony was picked and inoculated into 5 ml of kanamycin-positive (1:1000) LB liquid medium for overnight culture. The overnight culture solution was transferred to 1L kanamycin-positive (1:1000) LB liquid medium at a ratio of 1:100, and cultured with shaking at 200 rpm at 37°C for 3-4 hours until OD 60...

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Abstract

The invention discloses an enzymatic antibiotic for killing gram-positive bacteria and preparation and use of the enzymatic antibiotic. The enzymatic antibiotic is albumens consisting of an amino acid shown in SEQ ID NO.1. According to the invention, a gene with a special bacterium-splitting effect is screened from a streptococcus suis genome, and the enzymatic antibiotic which is a recombinant expression product with the bacterium-splitting activity is obtained by constructing a prokaryotic expression vector so as to determine the anti-bacterial activity, the bacterium-splitting spectrum and the bacterium-splitting efficiency as well as the optimal spitting condition which affects the activity of the expression product. The enzymatic antibiotic is strong in specificity, hardly causes resistance of bacteria and does not generate adverse influence on the host, so that the enzymatic antibiotic is a feasible method of solving bacterial drug resistance on the rise at present.

Description

technical field [0001] The invention belongs to the field of microbial chemistry, and relates to an enzyme antibiotic for killing Gram-positive bacteria and its preparation and application. Background technique [0002] Streptococcus suis is a zoonotic pathogen, which can cause human meningitis, piglet meningitis, sepsis, arthritis, endocarditis and other diseases, and severe cases can cause death. It has been found that there are more than 35 serotypes of the capsular antigen of this bacterium, and most of the pathogenic serotypes are 1-9. Among them, Streptococcus suis type 2 is the most common and most virulent serotype, and is the most popular. It is also the most pathogenic to pigs, causing huge economic losses to the pig industry, and in terms of public health, it poses a serious threat to the lives of relevant practitioners. Currently, the main treatment for S. suis is antibiotics. In recent years, due to the abuse of antibiotics in the breeding process, the drug re...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88C12N15/60C12N15/70C12N1/06A61K38/51A61P31/04
CPCC12N9/88A61K38/00C12N15/70
Inventor 严亚贤黄庆庆孙建和吉文汇
Owner SHANGHAI JIAO TONG UNIV
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