CN105543258B - A kind of preparation method and its antibacterial applications of Plesiomonas shigelloides bacterial virus catenase - Google Patents

Abstract

The invention relates to a preparation method of P. shigella-like phage lyase and its antibacterial application. Its amino acid sequence is Seq ID NO.2, which can prepare a drug for inhibiting bacterial contamination or overgrowth, and P. shigella-like phage lyase Bacteriophage lyases are used as bactericides. The enzyme finally lyses the host cell by hydrolyzing the amide bond between the sugar on the bacterial cell wall peptide sugar and the peptide or the bond between the amino acid residues in the peptide. The lyase not only acts specifically on the host, but also has a short action time and a wider action spectrum than that of phages. Lyase has the characteristics of high efficiency, specificity and no resistance, and has been successfully applied in the food and pharmaceutical industries.

Description

Preparation method and antibacterial application of a kind of P. shigella-like phage lyase

technical field

The design of the invention belongs to the field of bioengineering, and in particular relates to a P. shigella-like phage lyase (Endolysin) and its application as a bactericidal active component in medical hygiene, food processing and hygiene products.

Background technique

Phages are viruses that infect prokaryotic microorganisms such as bacteria. Mainly composed of nucleic acids and proteins. The genetic material of the phage is nucleic acid, and the protein constitutes the capsid of the phage. Phage has a simple structure and small size, and can pass through bacterial filters. It is a non-cellular microorganism that strictly parasitizes living cells. Phage synthesizes its own protein and nucleic acid by using the raw materials of the host bacteria, assembles in the host cell, lyses the cell, and releases the progeny phage.

With the improvement of people's living standards, people's demand for hand sanitizers is no longer just for cleaning, but also needs hand sanitizers to have the effects of sterilization, antibacterial and skin care. Now there are many antiseptic hand sanitizers on the market. However, the safety of its bactericidal ingredients is not high. After washing hands with low-toxic hand sanitizers, when hands directly touch food, the bactericidal ingredients will cause certain harm to people’s bodies, so their safety is not high. Other hand sanitizers are safe Although high in performance, its function is not complete, the preparation method is complicated, and the cost is high, which is unacceptable to general consumers. The invention patent of authorized notification number CN 101766543B is a chlorine dioxide-containing disinfectant hand sanitizer, and its bactericide is chlorine dioxide. Although chlorine dioxide has a good bactericidal effect, it is highly irritating. After contact, it mainly causes eye and respiratory tract irritation. Inhalation of high concentrations can cause pulmonary edema, which can be fatal. Patent CN101475879B a kind of sterilizing skin care hand sanitizer uses p-chloro-m-xylenol as a bactericide, which also has the disadvantages of strong irritation and toxicity. In order to overcome the shortcomings of chemical fungicides, the patent CN 101966134B, a bactericidal skin care hand sanitizer and its preparation method, discloses a hand sanitizer that uses paraben as a fungicide, but the solubility of paraben in water is extremely low, and the product is uniform poor stability. Invention patent CN 102600053B Chinese herbal medicine-inorganic antibacterial agent composite sterilizing hand sanitizer and its preparation uses the water extracts of mugwort leaves, artemisia annua, baicalin, pomegranate peel, and licorice as composite Chinese herbal antibacterial agents, and is used together with inorganic antibacterial agent nano-silver oxide. Complex, low antibacterial power, in order to prevent spoilage, it needs to be sterilized by autoclaving. Therefore, there is a great need for a hand sanitizer with good decontamination ability, strong bactericidal power, and safety and reliability on the market.

Contents of the invention

The purpose of the present invention is to overcome the deficiencies of the prior art, to provide a preparation method of P. shigella phage lyase and its antibacterial application. , good water solubility, broad antibacterial spectrum and other advantages.

The technical scheme that the present invention realizes purpose is as follows:

A gene coding for phage lyase of P. shigella-like bacteria, the nucleotide sequence of which is Seq ID NO.1.

A phage lyase from Pseudomonas shigella, the amino acid sequence of which is Seq ID NO.2.

Application of the P. shigella-like phage lyase in the preparation of medicines for preventing and treating bacterial infections, medical and sanitary products, food processing additives and sanitary products.

An engineering bacterium containing a gene encoding P. shigella phage lyase, the prokaryotic cell expression vector is pQE30, and the lyase gene is inserted between the KpnI and HindIII restriction sites of pQE30, The engineering bacterium is E.coli M15/pREP4/pQE30-gp2.

Moreover, the culture conditions of the engineering bacteria are: the culture temperature is 37°C, the inducer used for the induced expression is isopropyl-β-D-thiogalactopyranoside, the induction concentration is 0.5mmol/L, and the induced expression The temperature is 16°C.

A hand sanitizer containing phage lyase, the components and weight percentage are as follows: glycerin 1-5%, acetylated lanolin 1-3%, surfactant 8-30%, phage lyase 0.001-0.0001%, pH adjustment Agent 0-3%, essence 0.01-0.03%, and the rest is deionized water.

Advantage and positive effect of the present invention are:

This patent uses the phage lyase produced by the fermentation of genetically engineered strains as a bactericide, which has the advantages of safety, non-toxicity, good water solubility, and wide antibacterial spectrum. Lyase is a type of peptidoglycan hydrolase expressed by phage at the late stage of infection of the host, which finally lyses the host cell by hydrolyzing the amide bond between the sugar on the bacterial cell wall and the peptide or the bond between the amino acid residues in the peptide . The lyase not only acts specifically on the host, but also has a short action time and a wider action spectrum than that of phages. Lyase has the characteristics of high efficiency, specificity and no resistance, and has been successfully applied in the food and pharmaceutical industries.

Description of drawings

Figure 1 Construction and identification of the expression plasmid of the lyase gene gp2; lane 1: pQE30 vector BamHI digestion, lane 2: recombinant plasmid pQE30-gp2BamHI and HindIII double digestion verification, M: 5Kb DNA ladder;

Figure 2 Identification of lyase Gp2-induced expression; lane 1: total protein of uninduced HeL1, lane 2: total protein of HeL1 after induction with 0.5mmol/LIPTG for 4 hours, lane 3: precipitation after sonication, lane 4: supernatant after sonication , lane 5: purified lyase Gp2;

The lytic activity of Fig. 3 lyase Gp2 to Pseudomonas aeruginosa protoplast;

Fig. 4 The lytic activity of lyase Gp2 to free Pseudomonas aeruginosa.

Detailed ways

The method of the present invention is described below through specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be considered as illustrative rather than limiting the scope of the invention, the spirit and scope of which is defined only by the claims. For those skilled in the art, on the premise of not departing from the spirit and scope of the present invention, various changes or modifications to the material components and dosage in these embodiments also belong to the protection scope of the present invention.

Example 1

Extraction of phage genome

(1) Crude phage particles

(1) Preparation of host bacteria: Pick a single colony of Plesiomonas Shigelloides. ^[1] from the solid medium, inoculate it in 5 mL of LB liquid medium, and culture it with shaking at 37°C for 6-8 hours.

(2) Preparation of pure phage culture solution: pick a single phage plaque, inoculate it in 5mL logarithmic phase host bacteria culture solution, culture it with shaking at 37°C for 4-6h, then centrifuge the lysate at 10000rpm for 10min, and the supernatant is ready For the pure culture medium of bacteriophage.

(3) Preparation of crude phage particles: transfer the overnight culture of P. shigellai to 100 mL of liquid LB medium with an inoculum size of 1%, and amplify to the logarithmic phase (OD ₆₀₀ about 0.4) , add 5 mL of P. shigella-like phage pure culture solution, shake culture at 37° C. for 6-8 hours to obtain phage lysate. Add DNase I and RNase A to the lysate to a final concentration of 5 μg/mL, mix well and let stand at 37°C for 1 h. Then add NaCl to a final concentration of 0.1 mol/L, mix and dissolve, then ice-bath for 1 h, and centrifuge at 12,000 rpm for 20 min. After the supernatant was transferred to another centrifuge tube, PEG6000 was added to a final concentration of 10% (w/v), fully shaken and dissolved, then left at 4°C overnight, centrifuged at 12,000 rpm for 20 min, and the supernatant was discarded. Resuspend the precipitate with 500 μL TM (0.05mol/LTris-HCl pH 7.5, 0.2% MgSO ₄ 7H ₂ O) solution, and repeatedly extract once with an equal volume of chloroform, centrifuge at 12,000 rpm for 10 min to remove PEG6000 in the resuspension , and finally the crude extract of phage particles was obtained.

(2) Preparation of phage genomic DNA

(1) Add DNase I and RNaseA to the purified phage particles at a final concentration of 1 μg/mL, and let stand at 37°C for 1 hour to degrade the remaining DNA or RNA of the host bacteria;

(2) Then add 1mol/L EDTA (pH8.0) to a final concentration of 50mmol/L to terminate the activity of DNase I and RNase A;

(3) Add proteinase K to a final concentration of 50 μg/mL, add SDS to a final concentration of 0.5%, mix well, and digest the protein at 56°C for 1 h;

(4) Add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) and mix evenly, centrifuge at 12000 rpm for 10 min, and collect the supernatant;

(5) Repeat step 4 3 times; collect supernatant, equal volume of chloroform, mix, 12000rpm, 10min, collect supernatant;

(6) Add 1/10 volume of 3mol/L NaAc and 2 volumes of pre-cooled 95% ethanol, mix well, 12000rpm, 10min, precipitate DNA;

(7) Add 70% ethanol (500 μL) to the precipitate, invert the capped centrifuge tube several times, and centrifuge at 12000 rpm for 5 min to recover DNA.

(8) Remove the supernatant, remove the alcohol droplets on the tube wall, dry the open centrifuge tube at room temperature for 10 min, and then resuspend the DNA with double distilled water.

Example 2

Cloning of lyase gene gp2 and construction of expression vector

(1) Design a pair of specific primers according to the sequence encoded by the gp2 gene:

gp2F: 5'-CGG GGTACC ATG CAA CCA TCG CGA GCG TG-3', see Seq ID NO.3; gp2R: 5'-CCC AAGCTT CTG GCG GCG GTG GAT TTT TG-3', see Seq ID NO.4; Using the phage genome DNA as a template, the gp2 gene was amplified with the above primers, electrophoresed on 1% agarose, and the size of the amplified fragment was identified.

(2) The PCR product was directly digested with restriction endonucleases KpnI and HindIII, 37 ° C water bath for 2 hours, 1% agarose electrophoresis, and the gel recovery kit was used to purify and recover the fragments, and the fragments were combined with KpnI and HindIII before The double digested and purified pQE30 vector was ligated overnight at 16°C, and transformed into E. coli M15/pREP4 competent the next day. The transformation mixture was added to an appropriate amount of LB medium, and incubated at 37°C for 20min. The mixture was then spread on an LB plate containing ampicillin (100 μg/ml) and kanamycin (25 μg/ml), and cultured overnight at 37° C.;

(3) Identification of positive clones Pick positive clones and inoculate them in LB liquid medium containing kanamycin (25 μg/mL) and ampicillin (100 μg/mL), culture them overnight at 37°C, and extract by alkaline cracking Plasmids were identified by digestion with KpnI and BamHI, and the pQE30 empty plasmid digested with BamHI was used as a control. The correctly identified plasmid was named pQE30-gp2, and the bacteria containing the positive recombinant plasmid was named HeL1.

The results are shown in Figure 1. After the recombinant plasmid pQE30-gp2 was digested with BamHI, the pET28a(+) carrier band and the gp2 gene band appeared at 3.7kb and 0.8kb respectively, and the sizes were in line with expectations, indicating that the construction was correct.

Example 3

Induced expression and purification of lyase gp2 protein

Inoculate the bacteria HeL1 containing the recombinant plasmid into the LB medium containing ampicillin (100 μg/mL) and kanamycin (25 μg/mL), and cultivate overnight at 37°C with shaking; the next day, transfer to In 100mL LB medium, shake culture at 37°C until the OD ₆₀₀ value is about 0.6, add IPTG to a final concentration of 0.5mmol/L, and induce for 16h at 16°C. Bacteria were collected, cells were disrupted by ultrasonic waves, centrifuged at 10,000 rpm/min at 4°C for 10 min, the supernatant was collected, and the supernatant was filtered through a 0.22 μm filter membrane, and the protein expression in the lysed supernatant was analyzed by SDS-PAGE. The filtered lysed supernatant was purified with a His affinity chromatography nickel column (GE Healthcare, Sweden), specifically according to the kit instructions, and the obtained protein was named lyase Gp2.

The results of SDS-PAGE analysis are shown in Figure 2. After the engineered bacteria HeL1 containing the recombinant plasmid was induced by IPTG, there was an induced protein band at the position of about 18kD in the supernatant, which was consistent with the expected size, thus indicating that the recombinant HeL1 constructed Correct, and the expressed lyase protein product Gp2 is a soluble protein.

Example 4

Lysis of Pseudomonas aeruginosa protoplasts by lyase Gp2

(1) Preparation of Pseudomonas aeruginosa protoplasts

The overnight cultured Pseudomonas aeruginosa PAK was transferred to 250 mL LB medium, cultivated to OD ₆₀₀ =0.6, and the culture was centrifuged at 4000×g for 15 min at 4°C to collect the bacteria. The cells were resuspended in 0.05mol/L Tris buffer saturated with chloroform, and shaken gently for 45min at room temperature. Centrifuge at 4000×g for 15 minutes at 4°C, discard the supernatant to collect protoplasts. The protoplasts were washed and resuspended with 10 mmol/L phosphate buffered saline, and finally the protoplasts were resuspended to OD ₆₀₀ =0.6-1.0.

(2) Lysis of protoplasts

Lyase with a final concentration of 1 μg/mL was mixed with protoplasts and added to the cell culture plate. At room temperature, use a full-wavelength microplate reader to measure the absorbance OD ₆₀₀ of each sample, and use 10 mmol/L phosphate buffer For comparison. The decrease rate of absorbance OD ₆₀₀ was faster than that of the negative control, indicating that the protoplasts were lysed.

Results: Lyase Gp2 could efficiently degrade the protoplasts of Pseudomonas aeruginosa PAK at a concentration of 1 μg/ml. When the reaction was carried out for about 1 hour, 50% of the protoplasts of PAK were degraded.

Example 5

Lysis of free Pseudomonas aeruginosa cells by lyase Gp2

The overnight cultured Pseudomonas aeruginosa PAK was transferred to 250 mL LB medium, cultured to OD ₆₀₀ =0.6, centrifuged at 4000×g for 15 min, and the bacterial cells were collected. The bacteria were washed and resuspended with 10 mmol/L phosphate buffer, and finally the protoplasts were resuspended to OD ₆₀₀ =0.6-1.0. Lyase with a final concentration of 1 μg/mL was mixed with free PAK cells in a cell culture plate. At room temperature, use a full-wavelength microplate reader to measure the absorbance OD ₆₀₀ of each sample, and use 10 mmol/L phosphate buffer liquid as a control. The absorbance OD ₆₀₀ decreased faster than the negative control, indicating that the free cells were lysed.

Results: The lyase Gp2 can efficiently degrade Pseudomonas aeruginosa PAK at a concentration of 1 μg/ml, and 50% of the protoplasts of PAK were degraded when the reaction was carried out for about 1 hour.

Example 6

Bactericidal activity of lyase Gp2 as an active ingredient in hand sanitizer

Contents of each component by weight percentage of hand sanitizer:

Add glycerin, acetylated lanolin, and lauryl amidopropyl betaine to deionized water, heat to 55-60°C, stir evenly, cool to room temperature, adjust pH to 8-9 with citric acid, and then add biocide to lyse the bacteriophage Enzymes, flavors, stirring, degassing, packaging.

Table 1 biological sterilization hand sanitizer (1% dilution) 1 hour bactericidal effect

strain Pseudomonas aeruginosa Bacillus subtilis Bacillus licheniformis Bacillus mycoides Sterilization rate (%) 99.98 90.00 50.00 50.00

In addition to Example 6, the present invention also provides the following hand sanitizer formula, which contains phage lyase, which has a killing effect on Gram-positive bacteria and Gram-negative bacteria; the mass percentage of each component is: glycerol 1 ～5%, acetylated lanolin 1～3%, surfactant 8～30%, phage lyase 0.001～0.0001%, pH regulator 0～3%, essence 0.01～0.03%, and the rest is deionized water; The surfactants are sodium lauryl sulfate, sodium lauryl sarcosinate, ammonium lauryl sulfate, sodium fatty alcohol polyoxyethylene ether sulfate, ammonium fatty alcohol polyoxyethylene ether sulfate, coconut amidopropyl Betaine, Lauramidopropyl Betaine, Cocamidopropylamine Oxide, Triton X-100, Tween 80, Tween 20, Myristyltrimethylammonium Chloride, Decyl One or more mixtures of trimethylammonium bromide.

The preparation method of the bactericidal hand sanitizer is as follows: adding glycerin, acetylated lanolin, and surfactant to deionized water, heating to 55-60°C, stirring evenly and dissolving, cooling to room temperature, and using pH regulator sodium hydroxide or sodium carbonate to adjust the pH to 8-9, then add biocidal agent bacteriophage lyase, essence, stir, degas, and pack.

The basic principles and main features of the present invention have been shown and described above. Those skilled in the industry should understand that the present invention is not limited by the above-mentioned embodiments. What are described in the above-mentioned embodiments and the description only illustrate the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention will also have Variations and improvements are possible which fall within the scope of the claimed invention.

Claims (3)

1. A gene encoding a phage lyase of P. shigella-like, characterized in that: its nucleotide sequence is SeqID NO.1. 2. A P. shigella-like phage lyase, characterized in that its amino acid sequence is Seq ID NO.2. 3. A hand sanitizer containing phage lysing enzyme as claimed in claim 2, characterized in that: components and percentages by weight are as follows: glycerol 1～5%, acetylated lanolin 1～3%, surfactant 8～ 30%, phage lyase 0.001-0.0001%, pH regulator 0-3%, essence 0.01-0.03%, and the rest is deionized water.