CN105112393A - Bacteriophage fusion lyase capable of lysing Escherichia coli - Google Patents

Abstract

The invention provides a phage fusion lyase capable of lysing Escherichia coli for killing pathogenic Escherichia coli; the invention also discloses a construction and preparation method of the fusion lyase. Design small peptide primers, clone the target gene Lyase by PCR, connect it to the yeast expression vector pPICAα-A, construct pPICAα-A-Lyase eukaryotic expression recombinant plasmid, transform the recombinant plasmid into yeast GS115 competent, and express fusion Lyase, after protein purification, the cleavage activity was determined by spot method, using Pseudomonas aeruginosa CMCC, Escherichia coli DH5α, Escherichia coli O78 and Staphylococcus aureus ATCC27853 as indicator strains, the results showed that fusion lyase had a very obvious effect on Escherichia coli O78 Cracking activity, the action time is continuously effective from 2h to 24h. The invention has the characteristics of strong specificity, high sensitivity and good stability, and has great significance and practical application value for the prevention, control and killing of Escherichia coli.

Description

A phage fusion lyase capable of lysing Escherichia coli

technical field

The invention provides a phage fusion lyase capable of lysing Escherichia coli for killing pathogenic Escherichia coli, and the invention also discloses a construction and preparation method of the fusion lyase.

Background technique

In recent decades, due to the widespread use of antibiotics in the livestock production process, a large number of "super bacteria" (multi-drug-resistant and highly pathogenic bacteria) have been produced. New antibiotics that have only been put on the market for more than ten years, such as new veterinary drugs such as florfenicol and cefquinome, have also been resistant to bacteria. Common bacterial diseases are now difficult to control. In 2011, "Super Escherichia coli" caused piglet diarrhea with high morbidity and fatality nationwide, resulting in a 20% drop in pig production that year and a sharp rise in pork prices. At the same time, the drug resistance of bacteria also seriously endangers public health security. According to reports, the untreatable colibacillosis in Germany in 2011 was also related to the drug resistance of bacteria. Therefore, it is of great practical significance to develop a preparation that can kill "super bacteria" without producing drug resistance.

Research on bacteriophage and its lytic enzyme preparations has attracted more and more attention from researchers. Now there are relatively mature preparations going to the market for clinical application. For example, the lytic enzyme preparation developed for G+ bacteria of in vitro wounds, digestive tract, and respiratory tract infections (phages and lytic enzyme preparations of Staphylococcus aureus and Streptococcus have been used in the treatment of burns and scalds abroad). Although there are very few reports on lyase preparations against G ^- bacteria, they are limited to research. These preparations also have other deficiencies at the same time, such as the specificity of the bacteriophage preying on the bacterium with regions and bacterial strains; These deficiencies limit the role and development of phage and its lyase preparations. But these information also provide us with an idea, as long as the lyase is allowed to pass through the outer membrane of G ^- bacteria and interact with peptidoglycan in the periplasmic space, the lyase may destroy the cell wall of G ^- bacteria.

Contents of the invention

The purpose of the invention is to disclose a fusion lyase capable of lysing Escherichia coli, which solves the problem that the current lyase cannot lyse pathogenic Escherichia coli.

The present invention also provides the construction and preparation method of the fusion lyase, which can be used as an alternative medicine.

The fusion lyase capable of lysing Escherichia coli of the present invention is through modifying the structure of the phage lyase, adding a hydrophobic polypeptide to the C-terminus of the lyase, and the peptide can change the potential difference of the outer membrane of Escherichia coli, thereby changing the extracellular The structure of the membrane increases the gap between the outer membrane proteins, and the lyase can enter the periplasmic space of E. coli, interact with the peptidoglycan of G ^- bacteria, destroy the wall of G ^- bacteria, and play a role in killing bacteria .

The preparation method of the fusion lyase that can crack Escherichia coli of the present invention is as follows:

The phage fusion lyase is constructed, and the lyase passes through the outer membrane of G ^- bacteria and acts on peptidoglycan in the periplasmic space, and the lyase can destroy the cell wall of G ^- bacteria. Design primers, clone the target gene by PCR, connect it to the yeast expression vector pPICAα-A, construct pPICAα-A-Lyase eukaryotic expression recombinant plasmid, and transform the recombinant plasmid into Pichia pastoris GS115 competent cells , expressed pPICAα-A-Lyase phage fusion lyase, after the protein was purified, the cleavage range was determined by the spot method, and Pseudomonas aeruginosa ATCC27853, Pseudomonas aeruginosa CMCC, Escherichia coli DH5α, Escherichia coli O78, Escherichia coli CC11 and golden yellow Staphylococcus ATCC27853 was used as an indicator strain, and the results showed that Lysep3-D8 had a very obvious lytic activity against Escherichia coli O78, and the action time lasted from 2h to 24h. The invention can control the drug resistance of highly pathogenic bacteria (Escherichia coli) and the rapid spread of drug resistance among bacteria through various channels. It has the characteristics of strong specificity, high sensitivity and good stability, and has great significance and practical application value for the control and killing of Escherichia coli.

Description of drawings

Figure 1 is the phage plaque pattern formed by highly lytic phages and the electron micrographs of phages

Figure 2 is a picture of the in vitro sterilization results of pPICAα-A-Lyase phage fusion lyase

Detailed ways

The following examples are intended to further illustrate, but not limit, the invention. Those skilled in the art can understand that any parallel changes and modifications to the present invention will fall within the scope of the pending claims of the present invention without departing from the spirit and principle of the present invention.

Example 1

Phage Isolation

1.1 Collection of Pollutants

Collect 1000ml of sewage from pig farms near Changchun respectively

1.2 Culture of bacteria

Take the pathogenic E.coli strains from pigs (it is better to take several strains of Escherichia coli, if possible, it is best to isolate multi-drug resistant and highly pathogenic E. LB plates were cultured upside down in a constant temperature incubator at 37°C overnight, and a single colony was picked and inoculated into a test tube filled with LB culture medium, and cultured in a shaker at 37°C (180rpm) for 16 hours to obtain a bacterial suspension.

1.3 Isolation and propagation of phage

Add 10 mL of 10-fold concentrated peptone water, and add pathogenic Escherichia coli bacterial liquid at one time, add 0.2 mL of each bacterial liquid, culture at 37 degrees for 24 hours, take 5 mL of the culture solution in a sterilized test tube, and bathe in 56 degrees water bath for 1 hour , and then centrifuged at 4000r/min for 30min, and the supernatant was filtered through a 0.22um microporous membrane, and the obtained filtrate was a phage mixture, that is, the initially isolated phage.

1.4 Isolation and identification of phage

The identification of bacteriophage: adopt two-plate plate method (two) double-layer agar medium: lower layer medium: liquid LB medium+1.5% agar powder; Upper layer medium: liquid LB medium+0.7% agar powder) measure, will The 1.5% lower layer culture medium is used as the bottom layer agar, placed at 4 degrees for standby, and placed at 37 degrees for about half an hour before use. Mix 0.1ml of the phage stock solution and the same amount of E. coli culture solution evenly, incubate at 37 degrees for 20 minutes, add the mixed solution to the melted and insulated upper medium, mix well, and quickly pour into the prepared lower layer On the culture medium, rotate the plate to make it evenly distributed. After solidification, incubate it upside down at 37 degrees for 10 hours, and observe whether there are phage plaques. If moth-eaten plaques are observed, it indicates the presence of lytic phages

1.4.1 Phage purification

Because the size and shape of the phage plaques isolated for the first time were different, further purification was required. The specific operation method is as follows: (1) Use a sterilized 20 μL pipette tip to pick out a large-diameter phage plaque, put it in a centrifuge tube filled with 1 mL of SM preservation solution, blow it several times, and then place it in a refrigerator at 4°C for 3 hours. Filter with a 0.22 μm filter membrane, dilute the filtrate 10-fold with SM preservation solution, and take the last 3 dilution gradients for single-plaque culture with the double-layer plate method. 2) After culturing for 4 to 6 hours, pick a single plaque with a relatively large diameter from the plate with an autoclaved 20 μL pipette tip, and place it in liquid LB containing the host bacteria solution (the bacteria content is about 109 CFU/mL) for culture In the base, blow and beat several times, and then put it in a constant temperature shaker at 37°C at 75rpm and shake it for 6h for a small amount of proliferation. (3) After the proliferation, the mixture was centrifuged at 8000rpm for 20min, and the bacterial fragments were taken out. The supernatant was filtered with a 0.22 μm microporous membrane, and the morphology of the plaques was observed with the double-layer plate method on the filtrate. (4) After repeating the above operation 3 to 5 times, plaques of the same shape and size can be obtained. Pick a single plaque and place it in a centrifuge tube filled with 1mL of SM preservation solution, and store it in a refrigerator at 4°C (SM preservation Solution: take MgSO ₄ ·7H ₂ O2.0g, NaCl5.8g, 1MTris·C (lpH7.5) 50mL, 2% gelatin 5mL, add double distilled water to 1000mL, autoclave at 121°C for 20min, store at 4°C.)

1.4.2 Preservation of phages

For short-term storage at 4°C, and for long-term storage, centrifuge the phage proliferation solution at 5,000g for 10 minutes, collect the supernatant containing phage, and store it in two ways: (1) Add glycerol to the phage suspension to a final concentration of 30%, and divide into multiple tubes , stored at -70°C; (2) Divide the phage suspension into sterile EP tubes, add a drop of chloroform to each tube, and store at 4°C.

1.4.3 Liquid propagation of bacteriophage

The titer of the purified phage is generally not high, and further proliferation is required. In this experiment, we use the liquid proliferation method for proliferation. The specific method is as follows: add the phage liquid in a certain proportion to the host bacterial liquid cultured for 12 hours (logarithmic growth phase), then shake and culture at 37°C for 10 hours, centrifuge the mixed solution at 8000rpm for 15 minutes, remove the bacterial debris, and use the supernatant Filter through a 0.22 μm filter, then add the phage-containing filtrate to the host bacterial liquid cultured for 12 hours according to the original proportion, then put it into 37 ° C for 10 hours, and repeat the operation 3 to 5 times to obtain the higher titer Phage fluid.

1.4.4 Determination of potency after proliferation

The phage titer indicates the number of all invasive phages per milliliter of sample, also known as the number of plaque-forming units (pfu). Dilute the proliferated phage solution 10 times to the 7th power of 10 with SM preservation solution, take 100 μL of each of the last three dilution gradients and mix with 100 μL of the host bacterial solution, incubate at 37°C for 20 minutes, add to 47°C for incubation Mix evenly in the upper layer medium, quickly pour it on the surface of the bottom layer medium, rotate the plate, so that the mixture is evenly spread on the plate, and then put it in an incubator at 37°C for 4-6 hours and then count the plaques. Three parallels were performed for each gradient, and the average value was taken to calculate the phage titer. Phage titer (pfu/ml) = average number of plaques × dilution factor × 10

Determination of phage lysis profile: The single-spot method [40] was used to determine the lysis profile of phage. The bacterial strains used in this test were. Pick single colonies of bacteria and inoculate them in test tubes filled with 3 mL of LB, and incubate with shaking at 37°C for 8 hours to obtain bacterial liquids of each strain. Take 200 μL of bacterial suspensions and spread them evenly on ordinary agar plates, and take 10 μL of phage suspensions respectively. Drops are placed on different positions of the plate. When adding samples, there should be no contact between various phage suspensions, so as not to affect the test results. After natural drying, culture at 37°C for 6-8 hours, and observe the results.

Morphology. )

Example 2

whole gene sequencing

2.1 Enrichment of phage particles

Take a 50mL Erlenmeyer flask and add 10mL of liquid LB to sterilize it, then inoculate a single colony of the host bacteria, and culture it with shaking at 37°C overnight; take another 500mL Erlenmeyer flask and add 150mL of liquid LB to sterilize it, then inoculate 1mL for overnight culture The host bacterial liquid was shaken at 37°C until the initial logarithmic growth stage. Add phage according to the ratio of the multiplicity of infection to 0.1, let it stand at room temperature for 15 minutes, and continue shaking culture at 37°C for 6 hours; centrifuge the lysate at 4°C at 4000 rpm for 20 minutes, and centrifuge twice to remove bacterial debris, add DNaseI and RNaseA to the supernatant to a final concentration of 1ug/mL, stand at room temperature for 30 minutes; add NaCl to a final concentration of 1mol/L, stir and mix evenly, ice-bath for 1 hour, 4°C, 8000rpm centrifuge 10min, remove cell debris, collect supernatant, add NaCl to promote phage particles Separation from bacterial fragments is also necessary to effectively precipitate phage particles from polyethylene glycol; measure the supernatant, add PEG8000 at an amount of 10g per 100mL, gently invert to dissolve, do not violently, ice bath for more than 1 hour, and make the phage The particles form a precipitate; centrifuge at 10000rpm at 4°C for 10 minutes to recover the precipitated phage particles, discard the supernatant and invert the centrifuge tube for 5 minutes to drain the residual liquid, remove the residual liquid with a pipette; resuspend the precipitate with 2 mL of SM buffer; add etc. Volumetric chloroform extraction, slow shaking at 37°C for 30 seconds, centrifugation at 4,000 rpm for 15 minutes at 4°C, recovering the aqueous phase containing phage, and storing at 4°C;

2.2 Extraction of nucleic acid

Add DNase I to the final concentration of 10ug/mL and RNaseA to the final concentration of 5ug/mL to the purified phage particles, and incubate at 37°C for 1h to degrade the remaining DNA and RNA from the host bacteria; add EDTA (pH8.0) to the final Concentration 20mmol/Ml, inactivate DNase I;

Add proteinase K to a final concentration of 50ug/mL, SDS to a final concentration of 0.5%, mix well, 56°C

Incubate for 1 hour, then cool to room temperature; do not use equal amounts of saturated phenol, saturated phenol: chloroform: isoamyl alcohol (25:24:1), chloroform: isoamyl alcohol (24:1) to extract once each, centrifuge at room temperature at 5000rpm After 10 min, the upper aqueous phase was collected.

Take 2 times the volume of absolute ethanol from the upper aqueous phase, mix well, and place at -20°C overnight.

Precipitate phage nucleic acid; centrifuge at 12,000 rpm at 4°C for 20 min, discard the supernatant, wash the precipitate twice with 70% 4°C pre-cooled ethanol, and dry at room temperature; dissolve the precipitate with 50 μLTE (pH 8.0), and store at -20°C for later use.

2.3 Identification of phage nucleic acid type

Qualitative phage nucleic acid digestion by DNase and RNase:

Digest the phage nucleic acid with DNaseI, RnaseA and MungBeanNuclease routinely: mix the extracted nucleic acid with DNaseI (20U/ug), RnaseA (5U/ug) and MungBean (20U/ug) respectively, incubate at 37°C for 1h on 0.7% agar Sugar gel electrophoresis identification.

Phage nucleic acid was digested with restriction endonucleases EcoRI, BamHI and XbaI, respectively, at 37°C for 2 hours, and identified by 0.7% agarose gel electrophoresis.

2.4 Sequencing of recombinant plasmids constructed from phage DNA digested fragments

2.4.1 Double digestion and recovery of phage DNA

The phage DNA was digested with EcoRI and XbaⅠ, identified by 0.7% agarose gel electrophoresis, and the digested products were recovered with a gel recovery kit.

2.4.2 Digestion and recovery of pUC-18 vector

Digest the pUC-18 vector with EcoRI and XbaI, mix well, and digest at 37°C for 2 hours. Identify by 1% agarose gel electrophoresis, and recover the digested product with a gel recovery kit.

Example 3

Cloning and expression of pPICAα-A-Lyase fusion lyase gene and protein purification of pPICAα-A-Lyase

1. Materials and methods

(1) Strains and plasmids

Pichia pastoris GS115, Escherichia coli DH5a and pPICZα-A vectors were preserved by Room 5, No. 11 Institute of Military Medical Sciences, and pMD18-Tsimplevector was purchased from TakaRa Company.

(2) Related molecular biology operations

The extraction of total bacterial DNA, PCR amplification, plasmid recombination, Escherichia coli competent preparation, transformation, and plasmid extraction were carried out according to the methods described in "Molecular Cloning"; the recovery and purification of T4 ligase and DNA were carried out according to the kit instructions (purchased From TakaRa Company); DNA sequencing was entrusted to Shanghai Sangon Bioengineering Company.

(3) Amplification and cloning of phage fusion lyase gene

Design an upstream primer with a restriction endonuclease Xhol I site at the 5′ end:

5'-AGCTCTCGAGAAAAGAATGAAAATTTCATCCAATGGCCT-3' and

Downstream primer with restriction endonuclease EcoRI site

5'-CCGGAATTCTGCTCATATTATGGGAGCAGCAGCCCAAAATGCTGCCGCCACACCGCGTT-3'

The CFP10 and MPT64 genes were amplified using the genomic DNA of Mycobacterium tuberculosis S19 as a template. Reaction conditions: pre-denaturation at 95°C for 5 min, denaturation at 94°C for 1 min, annealing at 68°C for 1 min, extension at 72°C for 1 min, 30 cycles; final extension at 72°C for 10 min. The PCR products were ligated with pMD18-Tsimplevector to construct recombinant plasmids pMDCFP10 and MPT64.

(4) Construction of recombinant plasmid pPICAα-A-Lyase

Digest pMD18T-Lyase and pPICZα-A with restriction endonucleases XholⅠ and EcoRI, recover and purify Lyase target gene fragment and linearized pPICZα-A gene fragment through agarose gel. Ligate them with T4 ligase to form recombinant plasmid pPICAα-A-Lyase.

(5) SDS-PAGE analysis and purification of protein

The recombinant expression plasmid pPICAα-A-Lyase was linearized with the restriction endonuclease PmeI, electrotransformed into Pichia pastoris GS115 competent cells, and positive clones were picked and placed in YPD·zeocin and MD medium for 30 Shake culture at 180 rpm overnight, then inoculate into 5mL LB medium at a ratio of 1:100, culture at 37°C until OD ₆₀₀ =0.38, add methanol to a final concentration of 0.5%, induce expression for 24h, 48h, and 72h before harvesting. Centrifuge 1.5mL of the bacterial solution to collect the precipitate, add 50μL of sterilized water and 50μL of 2×SDS protein loading buffer, oscillate and mix well, boil in water for 15min, then centrifuge slightly, take 20μL for 12% SDS-PAGE analysis. The soluble protein was purified and recovered by histidine-binding resin column to obtain high-purity protein, and a single protein band could be seen in protein electrophoresis.

2. Results

SDS-PAGE detection of purified pPICAα-A-Lyase protein

The chromatographically purified CFP10 and MPT64 proteins showed a clear band by SDS-PAGE detection, and the purity was 96% by gel scanning.

Example 4

Evaluation and practice of cleavage activity of fusion lyase pPICAα-A-Lyase

When the lyase exists alone, like the PBS control, neither Escherichia coli nor Pseudomonas aeruginosa can be lysed; however, the lyase can significantly inhibit the growth of Escherichia coli and Pseudomonas aeruginosa; when the lyase is used in combination with citric acid, it can not only Escherichia coli can also effectively lyse Pseudomonas aeruginosa, and the action time lasts from 2h to 24h. Under the joint action of 0.5mg/mL final concentration of Lyase and 0.5mM final concentration of citric acid, the bacterial solution can be obviously changed from turbid to clear, the number of E. coli decreased by 7.2 times, Pseudomonas aeruginosa decreased by 17.3 times, and the number of bacteria in the blank control Respectively increased by 4.3 times and 14.3 times. Moreover, the effect of Lyase on Pseudomonas aeruginosa is stronger than that on Escherichia coli. This result fully proves that the Escherichia coli phage lyase is a potential broad-spectrum lyase, and there is an evolutionary correlation with the Pseudomonas aeruginosa phage lyase.

Claims (3)

1. an energy cracking colibacillary phage confluent lysis enzyme, is characterised in that: can break through intestinal bacteria adventitia, kill pathogenic colon bacillus.

2. the phage confluent lysis enzyme that claim 1 limits merges the little peptide that a section can change the outer membrane potential of intestinal bacteria.

3. the sequence of little peptide that claim 2 limits is:

The upstream primer of design 5 ' end band restricted property restriction endonuclease Xhol I restriction enzyme site:

5’-AGCTCTCGAGAAAAGAATGAAAATTTCATCCAATGGCCT-3’

With the downstream primer of restriction enzyme EcoRI restriction enzyme site

5’-CCGGAATTCTGCTCATATTATGGGAGCAGCAGCCCAAAATGCTGCCGCCACACCGCGTT-3’。