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Preparation method of porcine streptococcus phage perforin

A technology of Streptococcus suis and perforin, which is applied in botany equipment and methods, biochemical equipment and methods, and the use of vectors to introduce foreign genetic material, etc., can solve the problem of the increase of drug-resistant strains, the threat to the life safety of employees, and the economics of pig farming. Loss and other issues, to achieve the effect of improving cracking efficiency

Inactive Publication Date: 2010-09-15
SHANGHAI JIAO TONG UNIV
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Problems solved by technology

Streptococcus suis type 2 is the most prevalent and the most pathogenic to pigs, causing huge economic losses to the pig industry. In terms of public health, it poses a serious threat to the lives of relevant practitioners
For the treatment of streptococcosis suis is mainly antibiotics, however, with the widespread use of antimicrobials, the emergence of drug-resistant strains is further exacerbated

Method used

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  • Preparation method of porcine streptococcus phage perforin

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Embodiment

[0018] 1. Purification of phage, preparation of DNA and expression of perforin

[0019] Step 1: Take the phage SMP that grows on the plate as densely connected plaques (Ma, Y.L., Lu, C.P., Isolation and identification of a bacteriophage capable of infecting Streptococcus suis type 2 strains. Veterinary Microbiology, 2008, 132: 340-347, Whole Genome The sequence has been submitted to GenBank, accession number EF116926.), add 5mL SM (phage diluent, containing 5.8g NaCl, 2g MgSO4 7H2O, 50mL 1M Tris-Cl, pH 7.5, 5mL 2% gelatin per liter) to each plate, 4 ℃ Shake the shaker for 3 hours, collect the SM solution and put it in a sterile centrifuge tube for 10 minutes at 4000g to remove cell debris. Add pancreatic DNaseI and RNaseI to the supernatant to a final concentration of 1 μg / mL, and incubate at 37°C for 30 minutes. Add NaCl to a final concentration of 1 mol / L, and after 1 hour of ice-bathing, centrifuge at 11,000 g at 4°C for 10 minutes, and collect the supernatant. Add PEG 80...

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Abstract

The invention relates to a preparation method of porcine streptococcus phage perforin in the technical field of biological materials. The preparation method comprises the following steps of: (1) purifying to obtain a porcine streptococcus 2 type virulent phage and extracting the DNA of the phage; (2) amplifying perforin genes by applying a PCR (Polymerase Chain Reaction) through using the DNA obtained in the step (1) as a template; (3) carrying out prokaryocyte expression on the DNA obtained by amplifying in the step (2) to obtain fusion protein; (4) purifying the fusion protein and then carrying out renaturation to obtain the perforin. The purified and activated perforin can be used for cracking multiple porcine streptococcocci in vitro or improving the cracking efficiency of lyase.

Description

technical field [0001] The invention relates to a preparation method in the technical field of biological materials, in particular to a preparation method capable of lysing or enhancing lyase to lyse Streptococcus suis perforin (Holin). Background technique [0002] Streptococcus suis is a zoonotic infection that can cause meningitis, septicemia, arthritis, endocarditis, pneumonia in piglets and meningitis in humans. Streptococcus suis type 2 is the most prevalent and the most pathogenic to pigs, causing huge economic losses to the pig industry and posing a serious threat to the lives of relevant practitioners in terms of public health. The main treatment for S. suis is antibiotics. However, with the widespread use of antimicrobials, the emergence of drug-resistant strains has further aggravated. Perforin is a hydrophobic transmembrane protein encoded by the phage genome, which inserts into the cytoplasmic membrane at a specific stage to form non-specific holes or damages, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/33C12N15/70C07K14/005
Inventor 孙建和李宁严亚贤史一博陆承平
Owner SHANGHAI JIAO TONG UNIV
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