CN-115478064-B - Multi-target quorum sensing quenching enzyme preparation and preparation method and application thereof

CN115478064BCN 115478064 BCN115478064 BCN 115478064BCN-115478064-B

Abstract

The invention belongs to the technical field of quorum sensing quenching enzyme preparations, and particularly relates to a multi-target quorum sensing quenching enzyme preparation, a preparation method and application thereof, wherein the quorum sensing quenching enzyme preparation is mainly used for inhibiting pseudomonas aeruginosa virulence factors, the mass ratio of acyl homoserine lactone acyl transferase AiiO protein to 3-hydroxy-4-oxo quinolone 2, 4-dioxygenase AqdC protein is 20 (1-25), the quorum sensing quenching enzyme preparation is mainly used for inhibiting pseudomonas aeruginosa virulence factors and biomembrane synthesis, the mass ratio of deferiprone, aiiO, aqdC and deferiprone is 20 (1-25) (0.695-2.78), the prepared AqdC protein is high in yield and stability, the two enzymes are used in a combined mode after being used for regulating and controlling the dosage, the virulence factors regulated and controlled by different quorum sensing approaches in pseudomonas aeruginosa are effectively inhibited, and the quorum sensing quenching enzyme preparation is combined with the two enzymes to effectively inhibit pseudomonas aeruginosa and comprehensively inhibit the biomembrane.

Inventors

ZHAO JING
TIAN CHANG
YANG YAFEI
YANG JING
Liu Jiedai
QUAN CHUNSHAN
CHEN MING

Assignees

大连民族大学

Dates

Publication Date: 20260508
Application Date: 20220914

Claims (5)

1. The application of the multi-target quorum sensing quenching enzyme preparation in preparing a reagent for inhibiting virulence factors of pseudomonas aeruginosa is characterized in that the multi-target quorum sensing quenching enzyme preparation consists of acyl homoserine lactone acyltransferase AiiO protein and 3-hydroxy-4-oxo quinolone 2, 4-dioxygenase AqdC protein, wherein the mass ratio of the acyl homoserine lactone acyltransferase AiiO protein to the 3-hydroxy-4-oxo quinolone 2, 4-dioxygenase AqdC protein is 20 (1-25), and the virulence factors are extracellular total protease, pyocin and green fluorescein.
2. The application of the multi-target quorum sensing quenching enzyme preparation in preparing a reagent for inhibiting virulence factors and biological membranes of pseudomonas aeruginosa is characterized in that the multi-target quorum sensing quenching enzyme preparation consists of acyl homoserine lactone acyl transferase AiiO protein, 3-hydroxy-4-oxo quinolone 2, 4-dioxygenase AqdC protein and deferiprone, wherein the mass ratio of the three is 20 (1-25) (0.695-2.78), and the virulence factors are extracellular total protease, pyocin and green fluorescein.
3. The use according to claim 1 or 2, wherein, The AqdC protein is prepared by IPTG induction expression, and comprises the following steps: the plasmid pET28b (+):8-aqdC I is chemically or electrically converted into escherichia coli ESCHERICHIA COLI BL (DE 3), recombinant engineering bacteria E.coli BL21 (DE 3) -pET28b (+):8-aqdC I is utilized to carry out IPTG induction expression on the group induction quenching enzyme AqdC, 1% of the recombinant engineering bacteria is inoculated into LB liquid culture medium containing 50 mug/mL kanamycin at the final concentration, 10 h is activated under the condition of 37 ℃ and 180 rpm, 1% of the activated bacterial liquid is transferred into LB culture medium containing 50 mug/mL kanamycin at the final concentration, when the bacterial density value OD 600 reaches 0.6-0.8 under the condition of 37 ℃ and 180 rpm, IPTG with the final concentration of 0.2 mM is added to induce the target protein expression, and the recombinant engineering bacteria is cultured for 20-24 hours under the condition of 16 ℃ and 180 rpm, and the target protein is purified; The AqdC protein can also be prepared by a lactose self-induction method, and comprises the following steps: After activating E.coli BL21 (DE 3) -pET28b (+):8-aqdC I in 10mL liquid medium containing 50 mug/mL kanamycin, transferring the activated E.coli BL21 (DE 3) -pET28b (+):3%o inoculum size into 200mL lactose self-induction medium containing 50 mug/mL kanamycin, culturing under conditions that the liquid loading amount of lactose self-induction medium is 200mL,37 ℃ and 180 rpm until the cell density OD 600 reaches 0.6-0.8, cooling to 20-25 ℃ and culturing under conditions that the temperature is 250 rpm by using a baffle shake flask, and purifying target proteins.
4. The method according to claim 1, wherein the step of inhibiting virulence factors is performed by culturing Pseudomonas aeruginosa in 12-well plates for 8h hours, adding purified AqdC and AiiO protein solution to the plates, and adding 10-250 mu g AqdC protein and 200 mu g AiiO protein to each mL of culture solution for combination, and culturing to 24h hours.
5. The method of claim 2, wherein the step of inhibiting virulence factors and biofilm is performed by adding purified AqdC and AiiO protein solution and deferiprone solution to the well plate after culturing Pseudomonas aeruginosa in a 12-well plate for 8 h hours, and adding 10-250 mu g AqdC protein, 200 mu g AiiO protein and 50-200 mu M deferiprone solution to each mL of the culture solution for combination, and culturing to 24-h hours.

Description

Multi-target quorum sensing quenching enzyme preparation and preparation method and application thereof Technical Field The invention belongs to the technical field of quorum sensing quenching enzyme preparations, and particularly relates to a multi-target quorum sensing quenching enzyme preparation as well as a preparation method and application thereof. Background Quorum sensing refers to the phenomenon whereby microorganisms secrete specific signaling molecules and sense their concentration changes, thereby responding to changes in cell density. The quorum sensing system and the secreted signal molecules can participate in regulating and controlling the formation of a plurality of virulence factors and biological membranes of pathogenic bacteria, and are closely related to virulence and pathogenicity. It has now been found that there are a variety of quorum sensing systems in gram-negative pathogens whose corresponding signaling molecules are predominantly acyl homoserine lactones (N-acyl homoserine lactones, AHLs), quinolones (quinolones), etc., with different quorum sensing systems and signaling molecules being able to regulate different virulence factors. In addition, pathogenic bacteria are pathogenic and coexist, such as bacterial respiratory tract infection focus often exists a plurality of pathogenic bacteria which coexist, and intra-population and inter-population quorum sensing and information communication exist among the pathogenic bacteria, so that the pathogenicity of the quorum quenching enzyme can be exerted by inducing symbiotic bacteria through the signal molecules, and a better treatment effect can not be achieved when the quorum sensing system of a single kind in the pathogenic bacteria is inhibited. Pseudomonas aeruginosa is one of the most important conditional pathogenic bacteria causing infection of patients with burn injury, immunodeficiency or pulmonary fibrosis. Pseudomonas aeruginosa can cause host disease by secreting multiple virulence factors such as pyocin, green fluorescein, extracellular protease, and biofilm formation. Such as elastase, can cause tissue damage and inflammation, promoting invasion and colonization by pathogens. Pyocin destroys the host's defenses, causing pulmonary fibrosis. Green fluorescein is an important siderophore of pseudomonas aeruginosa, regulates secretion and production of toxins and damages the iron environment of a host. At present, due to the wide use of broad-spectrum antibiotics and extremely easy generation of drug resistance of pseudomonas aeruginosa, great difficulty is brought to clinical control of pseudomonas aeruginosa infection. Therefore, the development of a novel bacteriostat for pseudomonas aeruginosa and coexisting flora thereof has important significance for preventing and controlling pseudomonas aeruginosa and coexisting flora infection clinically. The pseudomonas aeruginosa is provided with a complex quorum sensing cascade regulation and control system, the Las quorum sensing system and the Rhl quorum sensing system of the pseudomonas aeruginosa are respectively regulated and controlled by AHL signal molecules 3-oxo-C12-HSL and C4-HSL, the PQS quorum sensing system is regulated and controlled by signal molecules PQS, the Las quorum sensing system and the Rhl quorum sensing system mainly regulate and control the synthesis of virulence factors such as protease, pyocin and the like, and the PQS quorum sensing system mainly regulates and controls the formation of virulence factors such as green fluorescein and the like. The inhibition of the complex quorum sensing system of pseudomonas aeruginosa is an effective way for realizing the comprehensive inhibition of various virulence factors. In addition, since iron is an important nutrient for pathogenic bacteria, limiting the available iron ion concentration can also inhibit biofilm synthesis as an alternative strategy to antibiotic therapy. Existing patents and achievements only contain a single class of quorum sensing quenching enzymes, such as: (1) The result of the interference research of targeted specific AHL lactonase on lung pseudomonas aeruginosa infection adopts paraoxonase to degrade pseudomonas aeruginosa signal molecule 3-oxo-C12-HSL, and inhibits the quorum sensing system and pathogenicity of the strain. (2) The patent "an N-acyl homoserine lactonase and its medicine" uses single acyl homoserine lactonase AiiK to inhibit the biomembrane, virulence factor extracellular protease and pyocin of Pseudomonas aeruginosa. (3) The patent 'a quorum-quenching enzyme OLB-26 and a coding gene and application thereof' mainly provides an amino acid sequence of a fusion quorum-quenching enzyme and determines the enzyme activity characteristics thereof, and the fusion protein fuses two quorum-quenching enzymes AiiO and AiiA coding genes degrading different AHL signal molecules. (4) Chinese literature "colony induction quenching enzyme clone expression and its effect on Pseudomonas ae