CN106854247A - A kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella - Google Patents

Abstract

The invention belongs to animal organism pharmaceutical engineering field, specifically disclose a kind of preparation method of the bacterial virus catenase that can crack Escherichia coli and salmonella, by designing primer, PCR Successful amplifications go out the lyase gene of phage E CGD1, and are transferred to expression system acquisition recombinant bacterium.Recombinant bacterium is expressed with IPTG inductions, and cellular lysate is obtained the restructuring lyases of soluble phage E CGD1 after induction, and the restructuring lyases can crack many plants of Escherichia coli and salmonella, i.e., with fragmentation pattern wider.Additionally, restructuring lyases shows with PB synergistic result, the presence for recombinating lyases reduces the minimum inhibitory concentration of PB, i.e., both has collaboration fungistatic effect.Recombinate the identification of lyases lytic activity, and its fragmentation pattern wide identification, disclose the restructuring lyases of phage E CGD1 in the prevention and control of Escherichia coli and salmonella infection with potential application value.

Description

Preparation of a phage lyase capable of lysing Escherichia coli and Salmonella method

technical field

The invention relates to the technical field of animal biomedical engineering, and more specifically, relates to a preparation method of a phage lyase capable of lysing Escherichia coli and Salmonella.

Background technique

Escherichia coli ( Escherichia coli ) is not only a normal resident bacterium in human and animal intestines, but also an important pathogenic bacterium. Escherichia coli can be roughly divided into three categories: intestinal non-pathogenic commensal strains, enteropathogenic strains and extraintestinal pathogenic strains. As a common pathogenic bacteria in humans and animals, it can cause intestinal infection, urinary tract infection, systemic infection and other clinical infections. Extraintestinal pathogenic strains can cause severe extraintestinal infections, especially urinary tract infections, and play an important role in the spread of antibiotic resistance. Among the multidrug-resistant strains of Escherichia coli, the proportion of extra-intestinal pathogenic strains was the majority. The emergence of multi-drug resistance has added many difficulties to the prevention and control of E. coli infection.

Salmonella ( Salmonella ), a gram-negative enteric bacterium, is an important pathogen of food-borne diseases, which has caused significant economic losses worldwide and seriously threatened public health security. Salmonellosis caused by Salmonella infection is a foodborne zoonosis with clinical symptoms including gastroenteritis, bacteremia, typhoid fever, and focal infection; symptoms of gastroenteritis can range from mild abdominal discomfort to vomiting and dehydration, and even die. Humans often become infected with Salmonella through ingestion of contaminated food of animal origin, such as eggs contaminated with Salmonella. Globally, there are an estimated 93,800,000 gastroenteritis cases and 155,000 deaths each year caused by Salmonella infection, of which about 85% are caused by food transmission.

In the current situation of Escherichia coli drug resistance, the problem of β-lactam antibiotic resistance is particularly serious. The resistance mechanism of these strains is mainly due to the existence of β-lactamase, which can degrade β-lactam antibiotics. In recent years, new β-lactamases have appeared in pathogenic strains outside the intestinal tract, such as plasmid-mediated AmpC β-lactamase, broad-spectrum β-lactamase and carbapenemase, etc. The best-known new type of β-lactamase was first reported in 1983 and was named broad-spectrum β-lactamase. This type of enzyme can degrade penicillins, cephalosporins and monocyclic β-lactam antibiotics, but cannot degrade cephamycin and carbapenem antibiotics, and its activity can be inhibited by typical β-lactamase inhibitors inhibition. Carbapenem resistance is a new problem that has emerged recently, mainly caused by plasmid-encoded carbapenemase, and currently carbapenem resistance mainly occurs in E. coli strains in hospitals. Prevention and control poses no small problem.

The problem of Salmonella drug resistance is also becoming more and more serious, and multi-drug resistant strains emerge in endlessly, and drug resistance to erythromycin, ampicillin, co-trimoxazole, ciprofloxacin, etc. have been reported. The problem of drug resistance of Salmonella in large-scale pig farms is also serious. A researcher analyzed the drug resistance of Salmonella isolated from pig farm feces and found that the isolated strains were resistant to tetracycline, ciprofloxacin, enrofloxacin, and amoxicillin. Xilin, kanamycin, florfenicol and ampicillin showed high drug resistance. At present, the widespread prevalence of multidrug-resistant strains and the continuous emergence of resistance to clinically important antibiotics such as quinolones and third-generation cephalosporins have become emerging thorny problems worldwide. Therefore, the research and development of new antibacterial agents is extremely urgent.

Bacteriophages are widely found in the environment, and they are viruses that host microorganisms such as fungi and bacteria. Potent phages have high-efficiency bactericidal capabilities. At the end of the infection cycle, the phage synthesizes a phage lyase, which destroys the cell wall of the host, causing the host cell to unbalance the osmotic pressure and finally rupture to release the progeny phage. Phages and their lytic enzymes also have a killing effect on antibiotic-resistant bacteria. Through genetic engineering technology, a large number of phage lytic enzymes can be artificially synthesized. Therefore, phage and its lyase have great application prospects.

Contents of the invention

The technical problem to be solved by the present invention is to seek new antibacterial preparations that can simultaneously inhibit Escherichia coli and Salmonella, in order to reduce and replace the use of antibiotics, and provide recombinant Escherichia coli expressing the phage recombinant lyase TrxA-lysin that can lyse Escherichia coli and Salmonella The preparation method of BL21/pET-32a-lysin.

The second object of the present invention is to provide the recombinant lyase TrxA-lysin prepared by the above method.

The third object of the present invention is to provide the recombinant lyase TrxA-lysin for lysing Escherichia coli and Salmonella.

The purpose of the present invention is achieved through the following technical solutions:

A preparation method of a phage lyase capable of cracking Escherichia coli and Salmonella, comprising the following steps:

S1. Amplify the lysin gene of the bacteriophage ECGD1; connect with the plasmid, and transfer it into the genetically engineered bacteria to obtain the genetically engineered bacteria containing the lysin recombinant plasmid;

S2. Cultivate the genetically engineered bacteria containing the lysin recombinant plasmid until the OD value of the bacterial solution is 0.6 to 0.8, induce the genetically engineered bacteria containing the lysin recombinant plasmid with IPTG, and obtain the soluble recombinant protein TrxA-lysin; the lysin gene The sequence of is shown in SEQ ID NO:1.

Preferably, the lysin gene of S1 is amplified by primers P1 and P2, and the sequences of the primers P1 and P2 are shown in SEQ ID NO: 2 and SEQ ID NO: 3.

The pET-32a expression system has the following advantages: it belongs to the prokaryotic expression system and is suitable for the expression of phage lyase gene. It has a thioredoxin TrxA gene, which can increase the disulfide bond formation of foreign proteins and increase the soluble expression of target proteins.

As a specific embodiment, the present invention designs primers to obtain the phage lysin gene capable of lysing Escherichia coli and Salmonella by using PCR method. The gene was connected to the pET-32a expression plasmid to construct the recombinant expression plasmid pET-32a-lysin, and the recombinant protein TrxA-lysin was induced and expressed by ITPG.

Preferably, the conditions for IPTG induction in S2 are as follows: the final concentration of IPTG is 1 mM, the induction time is 2 hours, and the induction temperature is 37°C.

As a more preferred embodiment, S1 of the preparation method of the phage lyase of the present invention comprises the following steps:

(1) Design primers and use PCR technology to amplify the lysin gene of bacteriophage ECGD1 that can lyse E. coli and Salmonella.

(2) Link the obtained lysin gene to pET-32a and screen to obtain the recombinant plasmid pET-32a-lysin; use the heat shock transformation method to introduce the pET-32a-lysin recombinant plasmid into E. coli BL21 to obtain recombinant E. coli BL21/pET -32a-lysin.

Preferably, (1) the reaction program of the PCR is: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, extension at 72°C for 10 s, completing 30 cycles; extension at 72°C for 5 min.

Preferably, (2) the heat-shock transformation method is as follows: take Escherichia coli BL21 competent cells, add pET-32a-lysin recombinant plasmid, mix well and ice-bath for 30 min, heat-shock transformation at 42°C for 90 s, and ice-bath After 2 minutes, LB liquid medium was added to recover for 30 minutes, and positive transformants were screened on the plate.

Preferably, the preparation method of the phage lysing enzyme of the present invention further includes the step of purifying the soluble recombinant protein TrxA-lysin.

More preferably, the step of purifying the soluble recombinant protein TrxA-lysin is: centrifuging to collect the BL21/pET-32a-lysin bacterial cells after induced expression, washing, resuspending and filtering, and loading the chromatographic column with Ni NTA Beads Purification of recombinant protein TrxA-lysin.

The present invention also provides the phage lyase recombinant protein TrxA-lysin obtained by the above method.

The invention also provides the application of the phage lyase recombinant protein TrxA-lysin in lysing bacteria.

The present invention also provides the application of the phage lysing enzyme recombinant protein TrxA-lysin in the preparation of medicaments for preventing and/or treating bacterial infection.

Preferably, in the above application, the bacteria are Escherichia coli and/or Salmonella.

Compared with the prior art, the present invention has the following beneficial effects:

The invention provides a method for preparing recombinant Escherichia coli expressing phage ECGD1 lyase lysin gene. That is, the E.coli BL21/pET-32a Escherichia coli expression system was used to recombine and express the lysin gene of the phage ECGD1 lysing enzyme, and the formation of the disulfide bond of the target protein was increased through the thireroreductin TrxA and the optimization of the expression conditions successfully obtained high Active soluble protein, protein purification does not need to go through complicated denaturation and refolding process. At the same time, after freezing and thawing the induced Escherichia coli BL21/pET-32a-lysin suspension three times between -80°C and room temperature, the bacterial suspension became clear and viscous, that is, through simple freezing and thawing, it can Ruptures the cells and releases most of the protein of interest. The preparation of the crude extract of the recombinant lyase in the present invention can be obtained by simple freezing and thawing, which is not only easy to operate and low in cost, but also not easy to cause denaturation of the target protein, and is suitable for mass preparation of the lyase.

The prepared recombinant protein TrxA-lysin was used to lyse Escherichia coli and Salmonella strains pretreated with chloroform, and it was found that TrxA-lysin had a lytic effect on multiple Escherichia coli and Salmonella strains, which confirmed that TrxA-lysin could lyse Escherichia coli at the same time and Salmonella. At the same time, the wide cleavage spectrum of TrxA-lysin reveals its potential application value in the prevention and control of Escherichia coli and Salmonella infection.

Description of drawings

Figure 1 is an electron micrograph of bacteriophage ECGD1 (magnified 49,000 times).

Figure 2 is the PCR amplification result of the lysin gene of bacteriophage ECGD1: M is DL 2 000 DNA Marker; 1 is the negative control; 2 and 3 are the PCR amplification products of the lysin gene.

Figure 3 is the double enzyme digestion identification results of recombinant plasmid pET-32a-lysin: M1 is DL10000 DNA Marker; M2 is DL 5000 DNA Marker; 1 is recombinant plasmid pET-32a-lysin; 2 is recombinant plasmid pET-32a-lysin The results were identified by double enzyme digestion with Nco I and Hind III.

Figure 4 shows the purification results of the lyase recombinant protein TrxA-lysin: M is the standard molecular weight of the protein; 1 is the total bacterial protein induced by BL21/pET-32a for 4 h; 2 and 3 are the 2 h of induction by BL21/pET-32a-lysin 4 is the lyase recombinant protein purified by nickel column.

Figure 5 shows the lysing effect of the lysing enzyme recombinant protein TrxA-lysin on DH5α cells pretreated with chloroform.

detailed description

The present invention will be further elaborated below in conjunction with the accompanying drawings and specific embodiments of the specification. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. For the experimental methods not indicated in the following examples, the specific conditions are usually in accordance with the conventional conditions in this field or the conditions suggested by the manufacturer. Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art.

Example 1 Construction of Escherichia coli BL21 expression strain containing lysin gene

Include the following steps:

S1. Construction of recombinant plasmid pET-32a-lysin

S11. Acquisition of lysin gene: A phage (named ECGD1, which can lyse Escherichia coli and Salmonella at the same time) was isolated from domestic sewage. High-throughput sequencing and assembly and analysis of sequencing results.

Extraction of bacteriophage ECGD1 genome: take 600 μl of phage concentrate after filter sterilization, add DNase I and RNase A to a final concentration of 1 μg/ml, digest overnight at 37°C (to completely remove host bacterial nucleic acid), inactivate at 80°C 15 min (note: this temperature cannot inactivate RNase A); add 24 μl of 0.5 mol/L EDTA (final concentration 20 mmol/L) and 1.5 μl of 20 mg/mL proteinase K (final concentration 50 μg /mL), 30 μl of 10% SDS (final concentration 0.5%), bathed in water at 56°C for 1 h; add an equal volume of equilibrated phenol, shake gently for 1 min, centrifuge at 12 000 rpm for 5 min, transfer the upper aqueous phase to a new centrifuge tube Medium; add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1), shake gently for 1 min, centrifuge at 12 000 rpm for 5 min, transfer the upper aqueous phase to a new centrifuge tube; add an equal volume of isopropanol, - Place at 70°C for 3 h; centrifuge at 13,000 rpm for 20 min at 4°C, and slowly pour off the supernatant; add an equal volume of 75% ice ethanol, let stand for 1 min, centrifuge at 7,000 g for 1 min, slowly pour off the ethanol, and store at room temperature After 10 min, the ethanol was completely evaporated, and the DNA was dissolved with an appropriate amount of deionized water, and stored at -20°C.

The whole gene sequence of phage ECGD1 was uploaded to GenBank (sequence number: KU522583.1), and the lysin gene was obtained through sequence annotation and alignment, with a size of 501 bp. Design primers P1 and P2 according to the gene sequence, and insert a restriction endonuclease Nco I restriction site into the upstream primer P1, Nco I is a fusion expression restriction site containing the initiation codon ATG, at its 5' end Plus 4 protective bases CATG. A restriction endonuclease Hind III restriction site was inserted into the downstream primer P2, and three protective bases CCC were added to the 5' end of the downstream primer. The lysin gene sequence is shown in SEQ ID NO: 1; the synthetic P1 and P2 primer sequences are shown in SEQ ID NO: 2-3; 1 μL of the genome of bacteriophage ECGD1 was used as a template, and the high-fidelity DNA polymerase Prime STAR Max ( 2×) 25 μL, 1 μL each of 10 μM primers P1 and P2, made up to 50 μL with ddH ₂ O, the reaction program was pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 55°C for 10 s, and 72°C Extend for 10 s to complete 30 cycles; extend at 72°C for 5 min. After the PCR reaction was completed, the product was observed and recovered on a 1% agarose gel, and an amplified band with a size of about 500 bp was seen, which was consistent with the expected result (Figure 2).

S12. Construction of the recombinant plasmid pET-32a-lysin: The PCR product recovered in S11 was digested with Nco I and Hind III, and a band of about 500 bp was recovered from the gel; pET-32a empty The plasmid was digested with double enzymes, and a band with a size of about 5900 bp was recovered from the gel. Take 4 μL of the complete modified fragment of lysin gene recovered from double-digested gel and 1 μL of pET-32a empty plasmid recovered from double-digested gel, add 1 μL of T4 DNA ligase and 1 μL of 10 × buffer, and use ddH Make up to 10 μL with ₂ O, mix well and place at 16°C for overnight connection, transform the connection product into E.coli DH5α competent cells, and place in LB agar culture plate containing 100 μg/mL ampicillin (Ampicillin), 37 Cultivate overnight, and then pick a single colony. The colonies were inoculated in LB liquid medium containing 100 μg/mL ampicillin for overnight culture. For PCR identification, use the bacteria liquid to be tested as a template, add 10 μL of DNA polymerase 2×EsTaq Master Mix, 0.5 μL of primers P1 and P2, 0.5 μL of template, make up to 20 μL with ddH ₂ O, and the PCR reaction program is 95°C pre- Denaturation for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min. The PCR product was detected by 1% agarose gel electrophoresis, and an amplified band with a size of about 500 bp was seen, which was consistent with the expected result (Figure 1). Use the plasmid DNA extraction kit to extract the plasmid from the positive bacterial solution, and perform double enzyme digestion identification and sequence determination. After double enzyme digestion, two target bands of about 500 bp and 5900 bp appeared in electrophoresis (as shown in Figure 3) , and the sequencing identification results were consistent with expectations, that is, the recombinant plasmid pET-32a-lysin was obtained.

S2. Construction of Escherichia coli BL21 expression strain containing lysin gene

S21. Preparation of Escherichia coli BL21 competent cells: Streak the frozen E.coli BL21 on the LB plate medium for recovery, pick a single colony and expand it overnight in the LB liquid medium. According to the ratio of 1:100, the BL21 cultivated overnight was inoculated in 25 ml of fresh LB liquid medium, cultivated to the logarithmic phase, and the OD _600nm value was about 0.6. The culture solution was transferred to a centrifuge tube, placed on ice for 10 min, and centrifuged at 3000 g for 10 min at 4°C. Discard the supernatant, gently suspend the cells with 10 mL of pre-cooled 0.05 mol/L CaCl ₂ solution, place on ice for 15-30 min, and then centrifuge at 3000 g for 10 min at 4°C. Discard the supernatant, add 4 mL of pre-cooled 0.05 mol/L CaCl ₂ solution containing 15% glycerol, gently suspend the cells, and place on ice for a few minutes to form a competent cell suspension.

S22. Transformation of Escherichia coli BL21 and PCR identification of transformants: Take 50 µL E. coli BL21 competent cells, melt on ice bath, add 1 µL pET-32a-lysin recombinant plasmid, mix gently; place the above mixture in ice bath After 30 min, heat shock was placed at 42°C for 90 s, the mixture was ice-bathed for 3 min quickly, 100 µL of LB liquid medium was added, and cultured at 37°C, 140 rpm for 45 min. The cultured bacterial liquid was evenly spread on LB solid medium containing 100 μg/mL ampicillin (Ampicillin), cultured overnight at 37°C, and a single colony was picked. The colonies were inoculated in LB liquid medium containing 100 μg/mL ampicillin for overnight culture. For PCR identification, use the bacteria liquid to be tested as a template, add 10 μL of DNA polymerase 2×EsTaq Master Mix, 0.5 μL of primers P1 and P2, 0.5 μL of template, make up to 20 μL with ddH ₂ O, and the PCR reaction program is 95°C pre- Denaturation for 5 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 10 min. . The PCR products were detected by 1% agarose gel electrophoresis, and an amplified band with a size of about 500 bp was seen.

S3. Induced expression process of E.coli BL21/pET-32a-lysin recombinant expression bacteria containing lysin gene:

The first is to determine the optimal induction time. The BL21/pET-32a-lysin identified as positive by PCR was streaked and cultured overnight; a single colony was picked and inoculated in 3 mL LA liquid medium, and cultured at 37°C and 220 rpm for 10 h. Take out the bacterial liquid and put it in the refrigerator at 4°C overnight; inoculate the above bacterial liquid into 3 mL LA liquid medium according to the volume ratio of 1%, shake at 37°C and 220 rpm; when the OD600 is 0.6-0.8, add the final concentration of 1 mmol/L IPTG for inducible expression. Take the bacterial solution induced at different times, centrifuge at 12,000 rpm for 2 min, remove the supernatant, add 100 µL of 1×SDS loading buffer to the bacterial pellet, boil in boiling water for about 5 min, centrifuge at 12,000 rpm for 2 min, and take the supernatant for SDS -PAGE electrophoresis to detect the best induction time. At the same time, set up pET-32a empty plasmid induced expression bacterial solution in BL21 as a control. Before loading the sample, put the gel into a vertical electrophoresis tank, set the voltage to 80 V for pre-electrophoresis for 30 min to eliminate the influence of unpolymerized substances in the gel; after loading the sample, set the voltage to 80 V, and wait for bromophenol blue dye to Press into a horizontal straight line, and increase the voltage to 160 V when it is about to enter the separation gel; stop electrophoresis when the dye is close to the bottom of the gel. Remove the gel, stain with Coomassie Brilliant Blue staining solution for about 1 h on a horizontal shaker, and then decolorize with decolorization solution, replace the decolorization solution during the period, decolorize until the protein zone is clear, and take pictures of the gel with a gel imaging system .

The results showed that the optimal induction time was 2 h. Use the optimal induction time determined above to induce expression of the target protein. The induced bacteria were collected by centrifugation, resuspended in 1 mL PBS, repeatedly frozen and thawed between -80°C and room temperature three times, centrifuged at 10,000 rpm for 2 min at 4°C, the supernatant was taken, and an equal volume of 2 ×SDS loading buffer, boiled in boiling water for about 5 minutes, centrifuged at 12,000 rpm for 2 minutes, and the supernatant was taken for SDS-PAGE electrophoresis to detect the expression form of the recombinant protein. The results showed that the target protein was mainly expressed in soluble form.

S4. Purification process of recombinant protein TrxA-lysin: BL21/pET-32a-lysin bacterial cells after induced expression were collected by centrifugation, and washed twice with PBS. According to the cell: Lysis buffer = 1:10 (W/V), suspend the cell, freeze and thaw three times between -80°C and room temperature, centrifuge at 10,000 rpm for 20 min at 4°C, and take the supernatant. And filter with a 0.22 μm filter membrane, collect the filtrate for the purification of the target protein.

Load Ni NTA Beads into a suitable chromatographic column, and equilibrate the column with 5 times the column volume of Lysis buffer, so that the filler and the target protein are in the same buffer system to protect the protein. Add the sample to the well-balanced Ni NTABeads (ensure that the target protein is in full contact with Ni ²⁺ to improve the recovery rate of the target protein), collect the effluent, and use it for SDS-PAGE analysis of protein binding. Wash with 10-15 times the column volume of Wash buffer to remove non-specifically adsorbed impurities, and collect the wash solution. Use 5-10 times the column volume of Elution buffer to elute the column, and collect the eluate, that is, the target protein fraction. The purified protein was subsequently desalted and concentrated using a 10 kDa ultrafiltration centrifuge tube. The purification results are shown in Figure 4.

Example 2 Activity detection of recombinant protein TrxA-lysin

Include the following steps:

S1. Cleavage of chloroform-pretreated DH5α with recombinant protein TrxA-lysin: Inoculate the strain to be tested in LB medium at a ratio of 1%, culture overnight, add chloroform at a final concentration of 5% to the bacterial solution, and shake for 15 min , collected by centrifugation, washed twice with deionized water, and stored at -80°C for later use. Before measuring the activity, the bacterial pellet was resuspended with 50 mmol/L Tris-HCl (pH 8.2, containing 0.1% Triton X-100). Add 10 µL of lysing enzyme solution (recombinant protein TrxA-lysin solution) to 90 µL of bacterial suspension, let stand at room temperature until the lysis effect is obvious, and measure OD ₄₅₀ . The experiment was repeated three times, and Tris-HCl buffer was used as a negative control. Since phage ECGD1 can lyse Escherichia coli genetically engineered strain DH5α, DH5α was first used as the test strain to detect the cleavage activity of the recombinant lyase TrxA-lysin. The cleavage activity of the recombinant lyase TrxA-lysin was detected by the turbidity method pretreated with chloroform, and it was found that the bacterial solution quickly became clear under the action of TrxA-lysin, and the OD ₄₅₀ decreased by 0.79 within 10 min (Figure 5), indicating that the The TrxA-lysin expressed in the test has cleavage activity.

S2. Combined use of polymyxin B and recombinant protein TrxA-lysin to lyse live bacteria: The test was divided into two groups, including the synergistic effect group of polymyxin B and recombinant protein and the polymyxin B control group. The experimental treatment of the synergy group is roughly as follows: prepare LB containing polymyxin B with a final concentration of 2 ¹⁰ , 2 ⁹ , 2 ⁸ , 2 ⁷ ... 2 ^-4 , 2 ^-5 µg/mL by 2-fold dilution method Culture medium (3 mL for each gradient), add 100 µL of recombinant protein TrxA-lysin solution to each dilution gradient, then inoculate Escherichia coli DH5α at a ratio of 1%, and culture overnight at 37°C with shaking. The polymyxin B control group was treated the same as above, but no recombinant protein TrxA-lysin solution was added. The results showed that in the polymyxin B control group, the minimum inhibitory concentration of polymyxin B on DH5α was 2 µg/mL; The minimum inhibitory concentration for DH5α is 1 µg/mL. That is, the existence of the lyase TrxA-lysin reduces the minimum inhibitory concentration of polymyxin B, thus proving that the two have synergistic antibacterial effects.

S3. Determination of the cleavage profile of the recombinant protein TrxA-lysin: use the chloroform pretreatment method described in S1 to detect the lysing activity of the lysing enzyme recombinant protein on the test strain, and observe the bacterial lysis effect and its OD ₄₅₀ value change. The test strains include clinical isolates of E. coli and Salmonella, as well as genetically engineered strains of E. coli. The lysis value of the lyase to be tested = Δ450nm (reduced value of the test group) - Δ450nm (reduced value of the Tris-HCl control group). Relative cleavage activity = cleavage value of lyase to test strain/cleavage value of lyase to DH5α. Taking Escherichia coli DH5α as the cleavage positive standard, the lysis activity of the lyase on Escherichia coli and Salmonella was detected by the turbidity method pretreated with chloroform. The results showed that the lyase had lysis activity on E. Broad-spectrum bactericidal activity against Escherichia coli and Salmonella. According to the results of relative lytic activity determination, the lytic activity of lyase to Escherichia coli is generally higher than that of Salmonella.

SEQUENCE LISTING

<110> South China Agricultural University

<120> A preparation method of phage lyase capable of lysing Escherichia coli and Salmonella

<130>

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 501

<212>DNA

<213> lysin gene sequence

<400> 1

atggctaaag tagttgatgt gtttgatatg ttacgttttg atgaaggact aaagctaact 60

gtatatactg ataccgaagg gtattggacg gttggtattg gacaccttct gacaaaactt 120

aaagataaat cagaagctat acgcattctt gataacttag taggtagaaa aactaatggg 180

gttattactg aagcggaagc tagaaaatc tttgagggtg acgtaaagaa agcgatacaa 240

caaatccatt caagtaccat attatctcct atttacgata aagtaagtcc taatcgtaaa 300

atggctatta tcaatatggt atttcaaatg ggattgaaag gtgcagaatc tttcaaaaat 360

agcttgactt tagtgagtaa ttcatattat actcaagcct ctataaattt acgtaaaagt 420

aaatggtatc gccaaacgcc taatcgcgca gagcgtgtaa ttcaggtgct taaaactgga 480

acattagacg cttataacta a 501

<210> 2

<211> 26

<212>DNA

<213> Primer P1 sequence

<400> 2

catgccatgg ctaaagtagt tgatgt 26

<210> 3

<211> 29

<212>DNA

<213> Primer P2 sequence

<400> 3

cccaagcttt tagttataag cgtctaatg 29

Claims (8)

1. a preparation method of a phage lyase that can crack Escherichia coli and Salmonella, is characterized in that, comprises the following steps: S1. Amplify the lysin gene of the bacteriophage ECGD1; connect with the plasmid, and transfer it into the genetically engineered bacteria to obtain the genetically engineered bacteria containing the lysin recombinant plasmid; S2. Cultivate the genetically engineered bacteria containing the lysin recombinant plasmid until the OD value of the bacterial solution is 0.6 to 0.8, induce the genetically engineered bacteria containing the lysin recombinant plasmid with IPTG, and obtain the soluble recombinant protein TrxA-lysin; the lysin gene The sequence of is shown in SEQ ID NO:1. 2. The preparation method of phage lysing enzyme according to claim 1, characterized in that, the lysin gene of said lysing enzyme of S1 is obtained by amplification of primers P1 and P2, and the sequence of said primers P1 and P2 is as SEQ ID NO: 2 and SEQ ID NO:3. 3. The preparation method of phage lyase according to claim 1, characterized in that the conditions for IPTG induction in S2 are: the final concentration of IPTG is 1 mM, the induction time is 2 hours, and the induction temperature is 37°C. 4. The preparation method of phage lysing enzyme according to claim 1, further comprising the step of purifying the soluble recombinant protein TrxA-lysin. 5. The phage lyase recombinant protein TrxA-lysin obtained by the method according to any one of claims 1 to 4. 6. The application of the phage lyase recombinant protein TrxA-lysin according to claim 5 in lysing bacteria. 7. The application of the phage lyase recombinant protein TrxA-lysin according to claim 5 in the preparation of a medicament for preventing and/or treating bacterial infection. 8. The application according to claim 6 or 7, characterized in that the bacteria are Escherichia coli and/or Salmonella.