Method for preparing enzyme of dissolving staphylococcal bacteria, its derivative, and method for preparing the derivative

A lysostaphinase and bacteria breaking technology, applied in the biological field, can solve the problems of high cost of PEG modification, the yield of lysostaphinase cannot meet large-scale preparation and the like, and achieves simple expression scheme, reduced immunogenicity and high yield Effect

Active Publication Date: 2007-12-26
HANGZHOU LION BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] The yield of expressing lysostaphin in the prior art cannot meet the requirements of large-scale preparation, and the cost of PEG modification is very high, so it is necessary to develop a method suitable for large-scale preparation of lysostaphin and a low-cost modification technology

Method used

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  • Method for preparing enzyme of dissolving staphylococcal bacteria, its derivative, and method for preparing the derivative
  • Method for preparing enzyme of dissolving staphylococcal bacteria, its derivative, and method for preparing the derivative
  • Method for preparing enzyme of dissolving staphylococcal bacteria, its derivative, and method for preparing the derivative

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Experimental program
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Effect test

Embodiment 1

[0051] Synthesis, cloning and expression of embodiment 1 lysostaphin gene

[0052] The expression vector is pET-22b (Invitrogen), and the host bacteria is Escherichia coli BL21 (DE3).

[0053] According to the Lysostaphin gene sequence (AX828346) of Staphylococcus simulans published by Genebank and the lysostaphin gene sequence of patent WO03074689, the lysostaphin gene sequence was optimally designed according to the needs of high-efficiency expression in Escherichia coli, see SEQ ID NO.1, gene encoding product See SEQ ID NO.2 for the amino acid sequence. Synthesize 20 complementary oligonucleotides, and introduce enzyme cutting sites Nde I and BamH I at the 5' end and 3' end respectively, according to the conventional method of molecular cloning, first treat with T4 phage polynucleotide kinase at 37°C After 30 minutes, the phosphorylated oligonucleotide fragments were mixed in equimolar ratios, denatured at 94°C for 5 minutes, annealed at 65°C for 10 minutes immediately, th...

Embodiment 2

[0059] Embodiment 2, the impact of different culture conditions on expression

[0060] The influence of different temperature conditions on the expression state of the product was studied by using medium (0.5% Yeast Extract, 1.0% Peptone, 0.5% NaCl, 0.05mg / L Amp, pH7.4). Inoculum size: 5% (the ratio of the volume of the seed liquid to the volume of the culture medium), the culture temperature before induction is 37° C., and the induction is carried out with IPTG, and the dose is 1 mmol / L. Induction was carried out at 4 temperatures after cultivation, and the results are shown in the table below.

[0061] induction temperature

[0062] Summary: After adding the inducer and culturing at 25°C for 4 hours, the expression level can reach 30%, and the target protein is mainly expressed in the supernatant of the broken bacteria. In the subsequent precipitation, the preferred condition is to induce expression at 25°C for 4 hours after culturing at 37°C. The target protein i...

Embodiment 3

[0063] Example 3 Separation and purification of expression products

[0064] Suspend the wet bacterial cells in a ratio of 20 ml / g with bacterial cell disruption buffer (40 mM Tris-Cl, 02 M NaCl, 0.5 mM EDTA, pH 8.0). Bacteria were disrupted by ultrasonic method (220V, 20khz, 900W, sonicated in an ice-water bath).

[0065] The bacteriostasis solution was centrifuged by high-speed centrifugation (10000g, 30min, 4°C), the precipitate was removed, and the supernatant was collected. The collected bacterial supernatant was separated and purified by ion exchange chromatography, and the process was to dilute with 20mmol / L Tris.CL buffer solution with a pH value of 8.5 to make the conductivity less than 5mS / cm. Use the same buffer solution to equilibrate the chromatographic column (50mm×10cm), and the chromatographic gel can be selected from CM-Sepharose Fast Flow, SP-Sepharose Fast Flow, Mono-S (Amersham Pharmacia). After sample loading, wash with equilibration buffer to the basel...

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Abstract

This invention relates to a method for preparing lysostaphin and its derivatives. The method comprises: designing and synthesizing lysostaphin gene, inserting lysostaphin gene into the digestion site of expression vector (plasmid pET), transforming E. coli, screening positive clones, culturing, inducing the expression of target protein, collecting E. coli, lysing, centrifuging, collecting the supernatant, and purifying the supernatant through chromatography. The method has such advantages as simple process, high expression level, simple purification, high yield, soluble product, and no requirement for renaturation, and is suitable for mass production. The lysostaphin derivatives are obtained by modifying lysostaphin with polyethylene glycol, and have such advantages as low immunogenicity and long half value period. The lysostaphin and its derivatives are suitable for treating Staphylococcus infection in clinic.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of lysostaphin and its derivative and the preparation method of the derivative. Background technique [0002] Staphylococcus (Staphylococcus) is a kind of Gram-positive coccus, which is widely distributed in nature, such as air, water, soil, feed and some articles, and also exists on the body surface, nasopharynx and intestinal tract of humans and animals. Most of them are not pathogenic, and a few can cause human and animal pathogenicity. Among them, Staphylococcus aureus is the most pathogenic, often causing purulent inflammation of the skin, tissues and organs, and is the cause of burn wound infection, acute liver failure and blood infection. The main pathogenic bacteria of diseases such as primary nephritis. Staphylococcus aureus also occurs simultaneously with G-sepsis bacteria, acting synergistically, seriously threatening the lives of patients. Enterotox...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N9/36C12N15/56C12N1/21
Inventor 刘国安刘沐荣康经武
Owner HANGZHOU LION BIOTECH
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