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Bacteriostatic-activity enhanced streptococcus-suis bacteriophage elysin and preparing method thereof

A phage lyase, Streptococcus suis technology, applied in the field of Streptococcus suis phage lyase, to achieve the effects of rapid action, enhanced activity, and resistance to resistance

Active Publication Date: 2016-06-22
JIANGSU UNIV
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Problems solved by technology

[0006] After analyzing the existing literature, using the error-prone PCR method to randomly mutate the LySMP coding gene, the mutant product obtained enhanced cleavage activity through prokaryotic expression. Using this enzyme and this process to screen Streptococcus suis phage lyase has not been done in China. see the report

Method used

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  • Bacteriostatic-activity enhanced streptococcus-suis bacteriophage elysin and preparing method thereof

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Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1: Prokaryotic expression and purification of Elysin

[0024] (1) Construction of elysin expression vector:

[0025] According to the lyase sequence of SMP on GenBank (GenBank No. EF116926.2), the gene sequence encoding LySMP (GenBank No. ABK91888.1) was synthesized, and the primers P1 and P2 for error-prone PCR were designed at the 5′ and 3′ ends, respectively. Insert restriction endonucleases EcoRI and XhoI restriction site, the primer sequence is as follows:

[0026] P1: 5′-GAG GAATTC ATGACAATCAACAT-3′;

[0027] P2: 5′-GCG CTCGAG AACGAATAAACTAC-3′

[0028] The error-prone PCR amplification kit (Shanghai Boyao Biotechnology Co., Ltd., ready-to-use error-prone PCR kit V1.2) was used to amplify the coding gene sequence of LySMP, so that the base mutation frequency was controlled at <1%.

[0029] PCR reaction system:

[0030] Error-prone PCRMix, 10 x 5 μL

[0031] dNTP for error-prone PCR, 10×5μL

[0032] MnCl 2 ,5mM5μL

[0033] LySMP gene 2 μL

...

Embodiment 2

[0056] Embodiment 2: the determination of Elysin optimal pH and temperature

[0057] Operation method reference literature: WangY, SunJH, LuCP. Purified recombinant phagelysin LySMP: an extensive spectrum of lytic activity for Swine Streptococci[J].

[0058] Streptococcus suis SS2-4 was centrifuged, treated with 20mM pH4.0 sodium acetate buffer, 20mM pH5.2 sodium acetate buffer, 10mM pH6.8 phosphate buffer, 10mM pH7.2 phosphate buffer and 20mM Tris-ClpH8.5, Adjust the bacterial OD with the above buffers 600 1.0-1.2. Add the above bacterial suspension into a 96-well plate, 100 μL per well. 100 μL of 50 μg / mL purified lyase was added to a 96-well plate, and three replicates were made for each buffer, and PBS buffer was used as a blank control. React at 37°C for 30 minutes, and measure the OD of Streptococcus suis SS2-4 600 The lowest pH value is the optimum pH value.

[0059] Then act at 4°C, 18°C, 25°C, 37°C, and 42°C for 30 minutes, each temperature was repeated three tim...

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Abstract

The invention belongs to the technical field of biology, and relates to a bacteriostatic-activity enhanced streptococcus-suis bacteriophage elysin and a preparing method thereof.A lySMP coding area gene sequence serves as a template, and is subjected to random mutation through error-prone PCR, then PCR products are connected with pET-28a (+) to be subjected to induction expression in BL21, and elysin with activity 1.8 times that of LySMP is obtained with the 96-hole-plate screening method; the most suitable pH and the most suitable temperature of the elysin are measured through a 96 hole plate.As the elysin has the advantages that acting is rapid, resistance is not prone to generation, and the elysin has the synergic sterilization effect with other antibiotics, the elysin is a potential bactericide; the effect of the elysin as the effective bactericide on resisting to the streptococcus-suis infection can be further researched through the obtained elysin, and contribution can be made in the control aspect of streptococcus suis.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a Streptococcus suis phage lyase with enhanced antibacterial activity [0002] The preparation method thereof is specifically the preparation method of the Streptococcus suis phage lysing enzyme elysin, especially the bacteriostatic activity of the lysing enzyme to Streptococcus suis SS2-4 is 1.8 times that of LySMP. Background technique [0003] Streptococcus suis is a disease caused by a variety of Streptococcus suis, in which infection with Streptococcus suis serotype 2 can lead to meningitis, sepsis, arthritis, endocarditis, pneumonia and human infection in piglets. Meningitis poses a serious threat to public health, especially the life safety of corresponding practitioners. At this stage, for the treatment of Streptococcus suis, the effective way is to use antibiotics. However, the abuse of antibiotics can lead to the drug resistance of Streptococcus suis; the application of pha...

Claims

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Application Information

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IPC IPC(8): C12N9/88C12N15/60A61K38/51A61P31/04
CPCA61K38/00C12N9/88
Inventor 王琰杨世兴郑敏杜杉杉冷靖李禹昕
Owner JIANGSU UNIV
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