CN114774391A

CN114774391A - Bacteriophage lysin for resisting escherichia coli and application thereof

Info

Publication number: CN114774391A
Application number: CN202210232896.0A
Authority: CN
Inventors: 董心迎; 刘珮琪; 谢倩梅; 李伟业; 曹雪薇; 黄秀琴; 江金飞; 代绘琳; 罗开健; 冯赛祥
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2022-07-22
Anticipated expiration: 2042-03-09
Also published as: CN114774391B

Abstract

The invention discloses an anti-Escherichia coli phage endolysin and application thereof. The amino acid sequence of the phage endolysin is shown in SEQ ID NO. 1. The invention clones the gene (SEQ ID NO.2) of the phage endolysin into an expression vector to obtain a recombinant expression vector, then transfers the recombinant expression vector into a host strain, and obtains the phage endolysin protein through induced expression.

Description

Bacteriophage lysin for resisting escherichia coli and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-escherichia coli phage endolysin and application thereof.

Background

The infection of bacteria in the growth process of livestock and poultry is a rare thing. As a kind of digestive tract bacterium, colibacillus is easy to cause high morbidity and high mortality of animals, increases the treatment cost of the animals and also seriously affects the economic benefit of the breeding industry. Feed, drinking water, and transmission media, etc. may be sources of infection by escherichia coli, and escherichia coli is widely present in animal feathers, feces, house air, etc. Because of the diversity of serotypes of escherichia coli and the low cross-immunity effect among serotypes, the infection and carrying of escherichia coli cannot be prevented and reduced by adopting the vaccine. Therefore, antibiotics are often used for treating colibacillosis and can effectively control the disease, but the long-term large-scale use of antibiotics causes that the colibacillosis is very easy to generate drug resistance. It is not uncommon for antibiotics to fail to work on E.coli, and even threatens food hygiene and safety.

In order to avoid the adverse effects of abuse of antibiotics, scientists are also constantly trying to develop new antibacterial agents specific to animals and their alternatives, of which phage therapy is of great interest. Bacteriophages are viruses that use microorganisms such as bacteria and fungi as hosts and are widely distributed in the natural environment and in the human body. The bacteriophage therapy is different from antibiotic therapy in that the bacteriophage therapy can specifically crack host bacteria, prevent and control specific bacteria and has no influence on other normal flora. The unique mechanism of action of bacteriophages has great advantages in avoiding bacterial resistance. In addition, the isolation of phages from nature also has advantages in terms of time and economic costs compared to the development of new antibiotics. In clinical practice, we have been able to eliminate pathogenic bacteria and ameliorate the corresponding conditions in patients or animals by means of bacteriophages.

Endolysin is a highly evolved peptidoglycan hydrolase coded by bacteriophage, is essentially a protein molecule, makes up the defect that the bacteriophage is essentially a virus, is more easily accepted clinically than the direct treatment of the bacteriophage, and is a novel antibacterial drug extracted from the bacteriophage. The endolysin is expressed in the process of bacteriophage replication, can destroy key chemical bonds of bacterial cell wall peptidoglycan, and as the peptidoglycan is the main structural component of the bacterial cell wall, the destruction of the peptidoglycan layer of the bacterial cell wall can cause the lysis of bacteria and the death of cells, so that progeny bacteriophage can be released to exert antibacterial activity.

Endolysins are emerging means of inhibiting pathogenic bacterial infections, and have the advantage of not being possessed by antibiotics. The phage endolysin is not easy to generate drug resistance, has wide sources, has good antibacterial effect on gram-positive bacteria in vivo and in vitro, and has good biological safety and higher species specificity. When endolysins are used as antibacterial agents, the outer membrane structure of gram-negative bacteria can prevent lytic enzymes from binding to the bacterial cell wall, resulting in difficulty in effective killing of gram-negative bacteria by endolysins. Therefore, the natural endolysin needs to be designed and modified by using a molecular biological method, so that the lytic activity and host spectrum of the endolysin are enhanced, and the endolysin becomes an excellent antibacterial agent.

Disclosure of Invention

The invention aims at overcoming the defects of the prior art and providing the phage endolysin for resisting the escherichia coli.

Another objective of the invention is to provide a method for preparing the phage endolysin resistant to Escherichia coli.

It is a further object of the present invention to provide the use of said bacteriophage lysin against E.coli.

The purpose of the invention is realized by the following technical scheme:

an anti-Escherichia coli phage endolysin, the amino acid sequence of which is shown in SEQ ID NO. 1.

The nucleotide sequence of the gene for coding the bacteriophage lysin for resisting the escherichia coli is shown as SEQ ID NO. 2.

A recombinant expression vector containing the gene coding the phage lysin against Escherichia coli is obtained by cloning the gene coding the phage lysin against Escherichia coli into an expression vector.

The expression vector is a PET series vector; preferably pET-9a, pET-28a (+), pET-22b (+), pET-26b (+) or pET-31b (+) vectors; further preferred is pET28a (+) vector.

The gene encoding the phage lysin described above against E.coli is cloned into an expression vector, preferably by insertion into pET28a (+) through the restriction sites HindIII and SalI.

A recombinant expression strain contains the recombinant expression vector, namely, the recombinant expression vector is transferred into a host strain to obtain the recombinant expression strain.

The host strain is bacteria, yeast or fungi; preferably bacteria; further preferred is Escherichia coli (Escherichia coli); most preferably E.coli BL21(DE 3).

A method for preparing phage endolysin (protein) resisting Escherichia coli comprises the following steps:

(1) cloning the gene of the phage endolysin for encoding the escherichia coli resistance into an expression vector to obtain a recombinant expression vector;

(2) transferring the recombinant expression vector into a host strain to obtain a recombinant expression strain;

(3) culturing the recombinant expression strain, inducing expression, centrifugally collecting bacterial liquid, separating and purifying to obtain the phage endolysin resisting colibacillus.

The expression vector in the step (1) is a pET series vector; preferably pET-3a, pET-9a, pET-28a (+), pET-22b (+), pET-26b (+) or pET-31b (+) vectors; further preferred is pET28a (+) vector.

The host strain in the step (2) is bacteria, yeast or fungi; preferably bacteria; further preferred is Escherichia coli (Escherichia coli); most preferably E.coli BL21(DE 3).

The culture conditions in the step (3) are as follows: culturing at 37 deg.C and 200rpm until OD600 of the bacterial liquid is 0.6-0.8 (preferably OD600 is 0.8).

The inducer for inducing expression in step (3) is preferably IPTG (isopropyl-. beta. -D-thiogalactopyranoside).

The dosage of the IPTG is calculated according to the addition of the IPTG in the induction system with the final concentration of 0.1 mmol/L.

The conditions for inducing expression in step (3) are preferably: inducing at 37 deg.C for more than 4 h.

The conditions for the centrifugation in step (3) are preferably: centrifuge at 8000rpm for 10 min.

The separation and purification in the step (3) is preferably performed by using a nickel column affinity chromatography.

The bacteriophage lysin for resisting escherichia coli is applied to preparation of a bacteriostatic agent (an antibacterial agent).

The bacteria in the bacteriostatic agent are gram-negative bacteria; preferably Escherichia coli; further preferred is pathogenic E.coli.

Compared with the prior art, the invention has the following advantages and effects:

(1) in view of the diversity of Escherichia coli serotypes and poor cross immunity among serotypes, the difficult function of vaccines and the emergence of drug-resistant strains of bacteria caused by the abuse of antibiotics, host bacteria can be cracked by phage therapy, the invention constructs a target gene, namely a gene for coding the phage endolysin protein resisting Escherichia coli, on an expression vector (preferably pET-28a (+)), carries out signal peptide deletion and other modification treatment on plasmids, ensures the smooth proceeding of transformation and protein expression, then transfers the recombinant expression vector into a host strain to induce and express to obtain the phage endolysin protein, and the phage endolysin protein has a certain bactericidal effect on Escherichia coli.

(2) The plasmid with the target gene constructed by the invention obtains a large amount of target protein through prokaryotic expression, is favorable for applying the phage endolysin to production and practice, effectively controls colibacillosis and ensures the safety of public health.

(3) The production method of the soluble anti-Escherichia coli phage endolysin protein provided by the invention is simple to operate, has high protein purity, good stability and biological activity, and has great significance for monitoring Escherichia coli.

(4) In order to comprehensively and accurately master the sterilization degree of the phage endolysin on escherichia coli, the invention monitors the sterilization effect of the phage endolysin under the environment of pH 6, detects the sterilization performance of the phage endolysin, and shows that the phage endolysin protein has certain antibacterial activity on escherichia coli.

Drawings

FIG. 1 is a diagram of a phage lysin protein recombinant expression vector against E.coli.

FIG. 2 is a diagram showing the results of PCR verification of the plasmid for expressing an E.coli-resistant phage lysin stored in example 2, transformed into the recipient E.coli BL21(DE3) (in the figure, lane 1 is Yeasen 2000 Marker; lane 2 is an amplified fragment of the target gene; and lane 3 is a negative control).

FIG. 3 is a graph showing the results of protein purification by SDS-PAGE electrophoresis (in the figure, lane 1 shows SMOBIO PM2510 Marker; and lane 2 shows the results of protein purification by IPTG 0.1mmol/L, which is an inducer, induced at 37 ℃ for 4 hours).

FIG. 4 is a graph showing the results of measurement of the activity of a phage endolysin protein at pH 6 (in the graph, the ordinate indicates the viable cell count of the 4 th well plate, the first bar indicates a blank control group (PBS) to which PBS was added, and the second bar indicates an experimental group (CJlysin) to which a phage endolysin protein resistant to E.coli was added).

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the invention are commercially available.

Example 1 construction of the anti-E.coli phage lysin CJlysin pET-28a (+)

According to the gene sequence (NCBI accession number: ERJ25909.1) of the anti-coliphage lysin, HindIII and SalI enzyme cutting sites are respectively added at two ends of a fusion gene (the gene sequence is shown as SEQ ID NO.2, and the coded protein is shown as SEQ ID NO.1) through gene synthesis, the fusion gene is connected with a pET-28a (+) vector which carries out double enzyme cutting of HindIII and SalI and is named as CJlysin pET-28a (+), the obtained nucleotide sequence of a connecting fragment is synthesized by Shanghai biological company, and the fusion gene with the size of 6041bp is obtained after the sequencing is correct.

Bacteriophage lysin amino acid sequence (SEQ ID NO. 1):

MNNFINSFNESKSITRVDKGMWVDGDKGVGVKADGDIRILRIRAFMCAIKYGEGTSGNNGYEINVGGKLFTKDYGKDFSDHPRYYVKSLDSSAAGAYQIKSSTWDMILKKYKKTYDITDFSPANQDKACLVLIKHIRNALDLIVDEKIDEAVRSRTDNDKKRLHYEWASMPDSPYGQRTITMEKFMEYYMHHLELEEIGISNLAISNKDIKKFLNGSHHHHHH*；

the phage lysin gene sequence (SEQ ID NO. 2):

ATGAACAACTTTATTAACTCTTTTAACGAATCCAAATCTATCACACGGGTGGATAAAGGCATGTGGGTCGATGGTGACAAAGGTGTTGGGGTGAAGGCCGATGGCGACATCCGTATTCTGCGTATTCGGGCGTTCATGTGCGCAATCAAATATGGGGAGGGCACTAGCGGCAACAACGGTTACGAAATCAACGTGGGCGGGAAATTATTTACCAAAGACTATGGTAAGGATTTTTCTGATCACCCACGGTATTACGTCAAAAGTCTCGACTCCTCAGCAGCTGGTGCATACCAGATTAAAAGTTCCACATGGGACATGATTCTGAAGAAGTATAAGAAAACTTACGATATTACCGATTTCTCACCAGCAAACCAGGATAAGGCTTGCCTTGTGCTGATTAAACACATTCGTAATGCACTTGACCTGATCGTGGACGAGAAGATTGATGAAGCCGTCCGGTCTCGTACCGATAATGATAAGAAACGCCTGCACTACGAATGGGCGTCAATGCCAGACTCTCCATATGGGCAACGTACTATTACTATGGAGAAGTTTATGGAATACTACATGCATCATTTGGAGCTCGAAGAGATTGGTATCTCGAACCTGGCCATCTCCAATAAAGACATCAAAAAATTCCTCAATGGTTCTCATCATCATCACCATCATTAA。

example 2 plasmid extraction, double restriction confirmation and sequencing

The synthetic plasmid (figure 1) of the Escherichia coli phage lysosome CJlysin pET-28a (+) resistant bacterium obtained in example 1 is transformed into recipient bacterium DH5 alpha, the bacterium solution is coated with LB (kanamycin (Kan) containing 50 mg/L) plate containing kanamycin for resuscitation and activation, after culturing for 16h at 37 ℃, a single colony is picked and is re-plated; selecting a part of the transferred bacterial colonies, transferring the selected bacterial colonies into a 100mL liquid LB culture medium, shaking the bacterial colonies on a shaker at 37 ℃ and 200rpm for 16 hours, temporarily storing the part of bacterial liquid at 4 ℃; and collecting a part of bacterial liquid by using a 50mL centrifuge tube, centrifuging at 8000rpm for 10min, discarding the supernatant, performing plasmid extraction, performing HindIII and SalI double enzyme digestion on the plasmid, performing nucleic acid electrophoresis to confirm the size of an enzyme digestion fragment, further sequencing the cloned plasmid of a band with the size of about 650bp, performing amplification culture on the positive clone with correct sequencing, extracting the plasmid, storing the plasmid to a refrigerator at the temperature of-20 ℃, and placing the bacterial liquid at the temperature of-80 ℃ by using LB solution containing 15-20% (v/v) glycerol for seed preservation.

Example 3 inducible expression of anti-E.coli phage lysin protein

(1) The plasmid expressing the phage lysin against E.coli, which was stored in example 2, was transformed into the recipient E.coli BL21(DE3), and after being placed on ice for 30min, it was heat-shocked in a water bath at 42 ℃ for 90s, then 1ml of LB broth was added, and after activation by thawing on a shaker at 37 ℃ and 220rpm, it was centrifuged at 6000rpm for 1min, 90% of the supernatant was discarded, and then the bacterial suspension was applied to LB plate containing kanamycin (kanamycin containing 30 mg/L) for activation by thawing, and after culturing at 37 ℃ for 16h, individual colonies were picked and subjected to colony PCR using T7 primers (forward primer T7-f and reverse primer T7-r), as shown in FIG. 2. Re-transferring the clone colony of the target band with the size of about 650bp to a coating plate and storing; wherein, the T7 primer sequence is shown as follows:

forward primer (t 7-f): 5'-TAATACGACTCACTATAGG-3', respectively;

reverse primer (t 7-r): 5'-TGCTAGTTATTGCTCAGCGG-3' are provided.

The PCR reaction system and conditions are as follows:

an amplification system:

PCR Master Mix enzyme 25. mu.L, forward primer (10 pmol/. mu.L) 1. mu.L, reverse primer (10 pmol/. mu.L) 1. mu.L, gene template 2. mu.L, ddH₂O make up to 50. mu.L.

And (3) amplification reaction conditions: 3min at 98 ℃; 40 cycles of 98 ℃ for 15s, 58 ℃ for 15s, and 72 ℃ for 60 s; 5min at 72 ℃.

After completion of the PCR reaction, electrophoresis was performed using 1% agarose gel. The gel electrophoresis showed the target band of about 650bp in size.

(2) Selecting a part of the transferred bacterial colonies, transferring the selected bacterial colonies into 100mL of liquid LB culture medium, and shaking the bacterial colonies on a shaker at 37 ℃ and 200 rpm; when the OD600 value of the bacterial liquid is 0.6, IPTG (isopropyl-beta-D-thiogalactoside) is used for induction, and the final concentration of the IPTG is 0.1 mmol/L; after induction for 4h, collecting the bacterial liquid by using a 50mL centrifuge tube, centrifuging at 8000rpm for 10min, and removing a supernatant;

(3) using imidazole-free protein lysate (NaH)₂PO₄·H₂6.9g (0.05 mol/L) of O (MW 137.99g/mol) and 17.54g (0.3 mol/L) of NaCl (MW 58.44g/mol), adding about 900mL of deionized water, stirring and dissolving, adding NaOH to adjust the pH value of the solution to 8.0, adding deionized water to reach 1000mL of constant volume, taking about 30mL of the solution, re-suspending the preserved bacteria solution, crushing by using an ultrasonic crusher, wherein the ultrasonic procedure is crushing for 3s, the interval is 5s, and the ultrasonic crushing is for 30 min;

(4) centrifuging the ultrasonically-crushed product at 8000rpm for 15min, collecting the supernatant, taking a small amount of the supernatant, adding 4 xSDS (sodium dodecyl sulfate) loading buffer solution, uniformly mixing, boiling in boiling water for 10min, if the heated sample has a viscous product, instantly centrifuging the sample, taking the supernatant, loading the supernatant into SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel holes purchased from Kinseri, simultaneously adding an equivalent amount of protein SMOBIO PM2510 Maker, adjusting to 100V with electrophoresis voltage, and running gel for 100 min. Taking out the gel block from the glass plate, and placing the gel block on a staining tank containing a Coomassie brilliant blue solution on a shaking table for 30 min; then pouring out the coomassie brilliant blue solution from the staining tank, adding clear water to wash the coomassie brilliant blue solution, putting the gel block into the staining tank in a shaking table, decoloring the gel block for 30min by using clear water to obtain a visible cleaning strip, and successfully expressing the solubility expression of the phage endolysin protein of the escherichia coli.

Example 4 purification of phage lysin protein against E.coli

Protein purification: the expressed phage endolysin protein resisting escherichia coli is purified into target protein by adopting a method of purifying prokaryotic expression by nickel column affinity chromatography protein in BIOSCIENCES company, and the purification steps are as follows:

(1) balancing: adding 1ml of nickel filler into a column special for purifying protein, and adding 10ml of lysine buffer prepared in advance (the formula of the lysine buffer is NaH)₂PO₄·H₂6.9g of O (MW 137.99g/mol), 17.54g of NaCl (MW 58.44g/mol) and 0.68g of imidazole (MW 68.08g/mol) are added into about 900mL of deionized water, stirred and dissolved, added with NaOH to adjust the pH value of the solution to 8.0, added with deionized water to reach the constant volume of 1000mL, and then the steps are repeated for 3-5 times.

(2) Sampling: adding the lysed bacteria liquid which is subjected to ultrasonication into the balanced column, and adding the lysed bacteria liquid in batches until the sample completely passes through the column.

(3) Washing: add prepared Wash buffer (6.9g NaH) to the column₂PO₄·H₂O (MW 137.99g/mol), NaCl (MW 58.44g/mol)17.54g, 1.36g imidazole (MW 68.08g/mol) added to about 900mL deionized water, after stirring to dissolve, NaOH was added to adjust the pH of the solution to 8.0, deionized water was added to bring to 1000mL), and the column was washed twice, 5mL each.

(4) And (3) elution: after the washing, an Elution buffer (Elution buffer solution: NaH) was added₂PO₄·H₂6.9g of O (MW 137.99g/mol), 17.54g of NaCl (MW 58.44g/mol) and 17.00g of imidazole (MW 68.08g/mol) are added into about 900mL of deionized water, stirred and dissolved, NaOH is added to adjust the pH value of the solution to 8.0, and deionized water is added to the solution to reach the volume of 1000mL) to obtain eggsThe white solution was eluted, 400. mu.l each, and washed 6 times.

(5) Collecting: each elution was labeled with a different sterilized 1.5ml EP tube and stored.

(6) Sample treatment: each eluted tube was subjected to SDS-PAGE by pipetting 10. mu.l of each sample (FIG. 3).

(7) And (3) storage: according to the results of SDS-PAGE, a sample having a high purity of the target protein was stored by adding 20% (v/v) of glycerol.

(8) BCA protein quantification: the protein was preserved by adding 20% (v/v) glycerol, and its protein concentration (2.6695mg/ml) was measured using BCA quantitative assay kit. Subpackaging the protein with the measured concentration, and storing at-20 ℃.

Example 5 detection of Activity of phage endolysin protein against E.coli

The activity of the phage lysin protein was measured at pH 6 by the following steps:

(1) escherichia coli strain BL21(DE3) was transferred to LB solid medium and cultured overnight at 37 ℃.

(2) A part of the colonies in step (1) was picked up and cultured on a shaker at 37 ℃ and 200rpm until OD600 became 1.

(3) Mu.l of the bacterial solution obtained in step (2) was diluted 10-fold in sterile PBS buffer (pH 6) in a 96-well plate⁴And (4) doubling. That is, 90. mu.l of sterile PBS buffer (pH 6) was added to 1-4 wells, 10. mu.l of the suspension obtained in step (2) was added to the 1 st well, and the pipetting was repeated 5 times, and 10. mu.l of the solution in the 1 st well was added to the 2 nd well, and the pipetting was repeated 5 times, and this was continued until the addition to the 4 th well.

(4) Mu.l of the 4 th well liquid obtained in step (3) was added to a blank well, and 5. mu.l of the protein purified and sterile filtered in example 4 (5. mu.l of sterile PBS buffer pH 6 was added to the blank), and the mixture was mixed by pipetting 5 times, covered with a lid, and incubated at 37 ℃ for 16 hours in an incubator.

(5) After the incubation is finished, all the liquid of the blank group and the experimental group are respectively taken out, the liquid is dripped on an LB culture medium, the culture medium is uniformly distributed with the liquid by rotating the culture medium, after the liquid is dried in the air, the liquid is placed in a 37 ℃ incubator for inverted culture overnight, whether the protein has antibacterial activity is judged by counting the number of bacterial colonies on the second day, the three times of the culture are repeated, and the result is shown in figure 4.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> south China university of agriculture

<120> bacteriophage lysin for resisting escherichia coli and application thereof

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<223> phage lysin

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Val Asp Lys Gly Met Trp Val Asp Gly Asp Lys Gly Val Gly Val Lys

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Ala Asp Gly Asp Ile Arg Ile Leu Arg Ile Arg Ala Phe Met Cys Ala

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Ile Lys Tyr Gly Glu Gly Thr Ser Gly Asn Asn Gly Tyr Glu Ile Asn

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Val Gly Gly Lys Leu Phe Thr Lys Asp Tyr Gly Lys Asp Phe Ser Asp

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His Pro Arg Tyr Tyr Val Lys Ser Leu Asp Ser Ser Ala Ala Gly Ala

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Tyr Gln Ile Lys Ser Ser Thr Trp Asp Met Ile Leu Lys Lys Tyr Lys

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Lys Thr Tyr Asp Ile Thr Asp Phe Ser Pro Ala Asn Gln Asp Lys Ala

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atgaacaact ttattaactc ttttaacgaa tccaaatcta tcacacgggt ggataaaggc 60

atgtgggtcg atggtgacaa aggtgttggg gtgaaggccg atggcgacat ccgtattctg 120

cgtattcggg cgttcatgtg cgcaatcaaa tatggggagg gcactagcgg caacaacggt 180

tacgaaatca acgtgggcgg gaaattattt accaaagact atggtaagga tttttctgat 240

cacccacggt attacgtcaa aagtctcgac tcctcagcag ctggtgcata ccagattaaa 300

agttccacat gggacatgat tctgaagaag tataagaaaa cttacgatat taccgatttc 360

tcaccagcaa accaggataa ggcttgcctt gtgctgatta aacacattcg taatgcactt 420

gacctgatcg tggacgagaa gattgatgaa gccgtccggt ctcgtaccga taatgataag 480

aaacgcctgc actacgaatg ggcgtcaatg ccagactctc catatgggca acgtactatt 540

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Claims

1. An anti-E.coli phage lysin, comprising: the amino acid sequence is shown in SEQ ID NO. 1.

2. A gene encoding the phage endolysin against Escherichia coli according to claim 1, characterized in that: the nucleotide sequence is shown in SEQ ID NO. 2.

3. A recombinant expression vector characterized by: comprising a gene encoding the phage endolysin against E.coli according to claim 1.

4. A recombinant expression strain characterized by: comprising the recombinant expression vector of claim 3.

5. A preparation method of phage lysin for resisting Escherichia coli is characterized by comprising the following steps:

(1) cloning a gene encoding the phage lysin of claim 1 into an expression vector to obtain a recombinant expression vector;

6. The method for producing an E.coli-resistant phage lysin according to claim 5, wherein:

the expression vector in the step (1) is a pET vector;

the host strain in the step (2) is bacteria, yeast or fungi;

the inducer for inducing expression in the step (3) is IPTG;

the dosage of the IPTG is calculated according to the addition of the IPTG in the induction system with the final concentration of 0.1 mmol/L;

the separation and purification in the step (3) is separation and purification by adopting a nickel column affinity chromatography.

7. The method for producing an E.coli-resistant phage lysin according to claim 6, wherein:

the expression vector in the step (1) is a pET-3a, pET-9a, pET-28a (+), pET-22b (+), pET-26b (+) or pET-31b (+) vector;

the host strain described in step (2) is Escherichia coli (Escherichia coli).

8. Use of an escherichia coli resistant phage lysin of claim 1 in the preparation of a bacteriostatic agent.

9. Use according to claim 8, characterized in that: the bacteria in the bacteriostatic agent are gram-negative bacteria.

10. Use according to claim 9, characterized in that: the bacteria in the bacteriostatic agent are pathogenic escherichia coli.