CN114774391A - Bacteriophage lysin for resisting escherichia coli and application thereof - Google Patents

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

A kind of anti-Escherichia coli phage endolysin and its application

technical field

The invention belongs to the field of biotechnology, in particular to a phage endolysin against Escherichia coli and its application.

Background technique

It is not uncommon for livestock and poultry to be infected with bacteria during the growth process. Escherichia coli, as a common bacteria in the digestive tract, can easily cause high morbidity and mortality in animals, increase the cost of animal treatment, and seriously affect the economic benefits of the breeding industry. Feed, drinking water, and transmission media may become sources of E. coli infection, and E. coli is widely present in animal feathers, feces, and house air. Due to the diversity of serotypes of Escherichia coli, and the low cross-immunity effect between serotypes, the use of vaccines cannot prevent and reduce the infection and carriage of Escherichia coli. Therefore, antibiotics are often used to treat colibacillosis and can effectively control the disease. However, the long-term use of antibiotics in large quantities has led to the emergence of drug resistance in Escherichia coli. It is not uncommon for antibiotics to fail to work against E. coli and even threaten food hygiene.

In order to avoid the adverse effects caused by the abuse of antibiotics, scientists are also constantly trying to develop new animal-specific antimicrobial drugs and their substitutes, among which bacteriophage therapy has attracted much attention. Bacteriophages are viruses that host microorganisms such as bacteria and fungi, and are widely distributed in the natural environment and the human body. Different from antibiotic therapy, phage therapy can specifically lyse host bacteria, prevent and control specific bacteria, and have no effect on other normal flora. The unique mechanism of action of bacteriophages has great advantages in avoiding bacterial resistance. In addition, isolating phages from nature has advantages in terms of time and economic cost compared to developing new antibiotics. In clinical practice, we have been able to eliminate pathogenic bacteria in patients or sick animals and improve the corresponding diseases through phages.

Endolysin is a highly evolved peptidoglycan hydrolase encoded by bacteriophage. It is a protein molecule in essence, which makes up for the deficiency that bacteriophage is a virus in nature. It is more clinically accepted than direct treatment of bacteriophage. a new class of antibacterial drugs. Endolysin is expressed during bacteriophage replication, and it can disrupt key chemical bonds in bacterial cell wall peptidoglycan. Since peptidoglycan is the main structural component of bacterial cell walls, disruption of the bacterial cell wall peptidoglycan layer will lead to bacterial lysis and cellular lysis. Death allows the release of progeny phage to exert antibacterial activity.

Endolysin has become an emerging means of inhibiting the infection of pathogenic strains, and its advantages lie in the advantages that antibiotics do not have. Phage endolysin is not easy to develop drug resistance, has a wide range of sources, has good antibacterial effect on Gram-positive bacteria in vivo and in vitro, has good biological safety and high species specificity. When endolysin is used as an antibacterial agent, the outer membrane structure of Gram-negative bacteria will hinder the binding of lyase to the bacterial cell wall, making it difficult for endolysin to effectively kill Gram-negative bacteria. Therefore, it is necessary to use molecular biology methods to design and transform natural endolysin to enhance the lytic activity and host spectrum of endolysin, making it an excellent antibacterial agent.

SUMMARY OF THE INVENTION

The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide an anti-Escherichia coli phage endolysin.

Another object of the present invention is to provide a method for preparing an anti-Escherichia coli phage endolysin.

Another object of the present invention is to provide the application of the described anti-Escherichia coli phage endolysin.

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

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 encoding the anti-Escherichia coli phage endolysin is shown in SEQ ID NO.2.

A recombinant expression vector containing the gene encoding the above-mentioned anti-Escherichia coli phage endolysin is obtained by cloning the above-mentioned gene encoding the above-mentioned anti-Escherichia coli phage endolysin into the expression vector.

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

The said gene encoding the above-mentioned anti-Escherichia coli phage endolysin is cloned into the expression vector, preferably inserted into pET28a(+) through the restriction sites HindIII and SalI.

A recombinant expression strain, which contains the above recombinant expression vector, is obtained by transferring the above recombinant expression vector into a host strain.

The host strain is bacteria, yeast or fungi; preferably bacteria; more preferably Escherichia coli; most preferably Escherichia coli BL21 (DE3).

A preparation method of an anti-Escherichia coli phage endolysin (protein), comprising the steps:

(1) clone the gene encoding the above-mentioned anti-Escherichia coli phage endolysin 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) firstly culture the recombinant expression strain, then induce expression, collect bacterial liquid by centrifugation, separate and purify, and obtain phage endolysin against Escherichia coli.

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

The host strain described in step (2) is bacteria, yeast or fungi; preferably bacteria; more preferably Escherichia coli; most preferably Escherichia coli BL21 (DE3).

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

The inducing agent for inducing expression described in step (3) is preferably IPTG (isopropyl-β-D-thiogalactoside).

The dosage of described IPTG is calculated according to its final concentration in the induction system being 0.1 mmol/L.

The conditions for inducing expression described in step (3) are preferably: induction at 37° C. for more than 4 hours.

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

The separation and purification described in step (3) is preferably carried out by nickel column affinity chromatography.

The application of the anti-Escherichia coli phage endolysin in the preparation of a bacteriostatic agent (antibacterial agent).

The bacteria in the bacteriostatic agent are Gram-negative bacteria; preferably Escherichia coli; more preferably pathogenic Escherichia coli.

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

(1) In view of the diversity of E. coli serotypes and the poor cross-immunity between serotypes, the vaccine is difficult to play, and the abuse of antibiotics leads to the emergence of drug-resistant strains of bacteria. The host bacteria can be lysed by phage therapy. The invention adopts the method of genetic engineering, constructs the target gene, that is, the gene encoding the anti-Escherichia coli phage endolysin protein, on the expression vector (preferably pET-28a(+)), and carries out the deletion of the signal peptide and other modification treatments on the plasmid, Ensure the smooth progress of transformation and protein expression, and then transfer the recombinant expression vector into a host strain to induce expression to obtain a bacteriophage endolysin protein, which has a certain bactericidal effect on Escherichia coli.

(2) The plasmid with the target gene constructed by the present invention can obtain a large amount of target protein through prokaryotic expression, which is beneficial to the application of the phage endolysin in production and practice, effectively controlling colibacillosis, and ensuring the safety of public health.

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

(4) In order to comprehensively and accurately grasp the bactericidal degree of the bacteriophage endolysin on Escherichia coli, the present invention monitors the bactericidal effect of the bacteriophage endolysin under the environment of pH=6, detects its bactericidal performance, and the results show that the bacteriophage Endolysin protein has certain antibacterial activity against Escherichia coli.

Description of drawings

FIG. 1 is a diagram of a recombinant expression vector for anti-Escherichia coli endolysin protein.

Fig. 2 is the PCR verification result of transforming the plasmid expressing anti-Escherichia coli phage endolysin stored in Example 2 into recipient Escherichia coli BL21 (DE3) (in the figure, lane 1 is Yeasen 2000Marker; lane 2 is Amplified fragment of target gene; lane 3 is negative control).

Figure 3 is the analysis result of protein purification detected by SDS-PAGE electrophoresis (in the figure, lane 1 is SMOBIO PM2510Marker; lane 2 is the result of protein purification induced by inducer IPTG 0.1mmol/L at 37°C for 4h).

Fig. 4 is a graph showing the detection result of phage endolysin protein activity under the condition of pH=6 (in the figure, the ordinate is the number of viable bacteria in the 4th well liquid drop plate, and the first bar graph represents the blank control of dripping PBS group (PBS), the second bar graph represents the experimental group (CJlysin) in which the anti-E. coli phage endolysin protein was added dropwise.

Detailed ways

The present invention will be described in further detail below with reference to the examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field. The test methods that do not specify specific experimental conditions in the following examples are usually in accordance with conventional experimental conditions or in accordance with experimental conditions suggested by the manufacturer. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

Example 1 Construction of anti-colibacillin endolysin CJlysin in pET-28a(+)

According to the gene sequence of anti-Escherichia coli endolysin (NCBI accession number: ERJ25909.1), the fusion gene (the gene sequence is shown in SEQ ID NO.2, and the encoded protein is shown in SEQ ID NO.1) is synthesized by gene synthesis ) were added with HindIII and SalI restriction sites, respectively, and were connected to the pET-28a(+) vector that was double-digested by HindIII and SalI, named CJlysin in pET-28a(+), and the obtained ligation fragment nucleotides The sequence was synthesized by Shanghai General Biological Company, and after correct sequencing, a fusion gene with a size of 6041 bp was obtained.

Phage endolysin amino acid sequence (SEQ ID NO. 1):

MNNFINSFNESKSITRVDKGMWVDGDKGVGVKADGDIRILRIRAFMCAIKYGEGTSGNNGYEINVGGKLFTKDYGKDFSDHPRYYVKSLDSSAAGAYQIKSSTWDMILKKYKKTYDITDFSPANQDKACLVLIKHIRNALDLIVDEKIDEAVRSRTDNDKKRLHYEWASMPDSPYGQRTITMEKFMEYYMHHLELEEIGISNLAISNKDIKKFLNGSHHHHH*;

Phage endolysin gene sequence (SEQ ID NO. 2):

ATGAACAACTTTATTAACTCTTTTAACGAATCCAAATCTATCACACGGGTGGATAAAGGCATGTGGGTCGATGGTGACAAAGGTGTTGGGGTGAAGGCCGATGGCGACATCCGTATTCTGCGTATTCGGGCGTTCATGTGCGCAATCAAATATGGGGAGGGCACTAGCGGCAACAACGGTTACGAAATCAACGTGGGCGGGAAATTATTTACCAAAGACTATGGTAAGGATTTTTCTGATCACCCACGGTATTACGTCAAAAGTCTCGACTCCTCAGCAGCTGGTGCATACCAGATTAAAAGTTCCACATGGGACATGATTCTGAAGAAGTATAAGAAAACTTACGATATTACCGATTTCTCACCAGCAAACCAGGATAAGGCTTGCCTTGTGCTGATTAAACACATTCGTAATGCACTTGACCTGATCGTGGACGAGAAGATTGATGAAGCCGTCCGGTCTCGTACCGATAATGATAAGAAACGCCTGCACTACGAATGGGCGTCAATGCCAGACTCTCCATATGGGCAACGTACTATTACTATGGAGAAGTTTATGGAATACTACATGCATCATTTGGAGCTCGAAGAGATTGGTATCTCGAACCTGGCCATCTCCAATAAAGACATCAAAAAATTCCTCAATGGTTCTCATCATCATCACCATCATTAA。

Example 2 Plasmid extraction, double digestion to confirm the plasmid and send for sequencing

The synthetic plasmid (Figure 1) obtained in Example 1 against Escherichia coli endolysin CJlysin in pET-28a (+) was transformed into the recipient bacteria DH5α, and the bacterial solution was coated with LB (containing 50 mg/L) containing kanamycin. After culturing at 37°C for 16h, pick a single colony to transfer to the plate again; pick a part of the transferred colony and transfer it to 100mL of liquid LB medium In the medium, shake the bacteria on a shaking table at 37 °C and 200 rpm for 16 h, first temporarily aliquot part of the bacterial liquid and store it at 4 °C; then take a part of the bacterial liquid for collection in a 50 mL centrifuge tube, centrifuge at 8000 rpm for 10 min, discard the supernatant, and carry out plasmid extraction. Extraction and double digestion of the plasmid with HindIII and SalI, run nucleic acid electrophoresis to confirm the size of the digestion fragment, and further sequence the cloned plasmid of the target band with a size of about 650bp, expand the positive clone with correct sequencing, and extract the plasmid for preservation To -20°C refrigerator, place the bacterial liquid in LB solution containing 15% to 20% (v/v) glycerol at -80°C for seed preservation.

Example 3 Induction and expression of anti-coliphage endolysin protein

(1) The plasmid expressing the anti-Escherichia coli phage endolysin stored in the above Example 2 was transformed into the recipient Escherichia coli BL21 (DE3), placed on ice for 30min, and then added after heat shock in a 42°C water bath for 90s 1ml of LB broth medium was revived and activated at 37°C on a shaking table at 220rpm, centrifuged at 6000rpm for 1min, discarded 90% of the supernatant, and the bacterial solution was coated with LB containing kanamycin (containing 30mg/L of kanamycin). T7 primers (forward primer t7-f and reverse primer t7-r) were used for colony PCR confirmation, as shown in Figure 2. The cloned colonies of the target band with a size of about 650bp were re-plated and saved; the T7 primer sequences were as follows:

Forward primer (t7-f): 5'-TAATACGACTCACTATAGG-3';

Reverse primer (t7-r): 5'-TGCTAGTTATTGCTCAGCGGG-3'.

The PCR reaction system and conditions are as follows:

PCR Master Mix酶25μL，正向引物(10pmol/μL)1μL，反向引物(10pmol/μL)1μL，基因模板2μL，ddH₂O补足至50μL。Amplification system:

PCR Master Mix enzyme 25 μL, forward primer (10 pmol/μL) 1 μL, reverse primer (10 pmol/μL) 1 μL, gene template 2 μL, ddH ₂ O supplemented to 50 μL.

Amplification reaction conditions: 98°C for 3 min; 98°C for 15s, 58°C for 15s, 72°C for 60s, 40 cycles; 72°C for 5 min.

After the PCR reaction was completed, 1% agarose gel was used for electrophoresis. Gel electrophoresis showed the target band with a size of about 650bp.

(2) with the colony after the transfer, pick a part of the colony and transfer it to the liquid LB medium of 100 mL, shake the bacteria on a shaking table at 37° C. and 200 rpm; when the OD600 value of the bacterial liquid is 0.6, use IPTG (isopropyl -β-D-thiogalactoside) was induced, and the final concentration of IPTG was 0.1 mmol/L; after 4 hours of induction, the bacterial liquid was collected in a 50 mL centrifuge tube, centrifuged at 8000 rpm for 10 min, and the supernatant was discarded;

(3) Lysis buffer (NaH ₂ PO ₄ ·H ₂ O (MW 137.99g/mol) 6.9g, i.e. 0.05mol/L, NaCl (MW 58.44g/mol) 17.54g, i.e. 0.3mol, without imidazole /L, add about 900mL of deionized water, after stirring to dissolve, add NaOH to adjust the pH value of the solution to 8.0, add deionized water to make up to 1000mL), take about 30mL of the stored bacterial solution to resuspend, and use an ultrasonic crusher for crushing , the ultrasonic program is broken for 3s, the interval is 5s, and the ultrasonic break is 30min;

(4) Centrifuge the sonicated product at 8000rpm for 15min, collect the supernatant, take a small amount of supernatant and add 4×SDS sample buffer, after mixing, boil in boiling water for 10min, if the heated sample has a viscous product, After the samples were centrifuged briefly, the supernatant was taken and loaded onto the wells of SDS-PAGE gels purchased from GenScript. At the same time, an equal amount of protein SMOBIO PM2510Maker was added, and the electrophoresis voltage was adjusted to 100V to run the gel for 100 min. Take the gel block out of the glass plate, and put it on the staining tank containing Coomassie brilliant blue solution on the shaker for 30 minutes; then pour out the Coomassie brilliant blue solution in the staining tank, add water to rinse it, and place the gel block on the shaker. In the staining tank in , decolorize the gel block with water for 30 minutes, and then the washed band can be seen, and the soluble expression of the anti-Escherichia coli phage endolysin protein was successfully expressed.

Example 4 Purification of phage endolysin protein against E. coli

Protein purification: The expressed anti-Escherichia coli phage endolysin protein is purified by the method of purifying prokaryotic expression protein by nickel column affinity chromatography in BIOSCIENCES company. The purification steps are as follows:

(1) Equilibrium: add 1ml of nickel filler to the column dedicated to purification of protein, add 10ml of Lysisbuffer prepared in advance (the formula of Lysis buffer buffer is: NaH ₂ PO ₄ ·H ₂ O (MW 137.99g/mol) 6.9g, NaCl (MW58.44g/mol) 17.54g, 0.68g imidazole (MW 68.08g/mol) add about 900mL deionized water, after stirring and dissolving, add NaOH to adjust the pH value of the solution to 8.0, add deionized water to make up to 1000mL) Carry out balanced packing and repeat 3 to 5 times.

(2) Sample loading: Add the sonicated lysate to the equilibrated column, and add it in batches until the sample completely passes through the column.

(3) Washing: Add the prepared Wash buffer (6.9g NaH ₂ PO ₄ ·H ₂ O (MW 137.99g/mol), NaCl (MW 58.44g/mol) 17.54g, 1.36g imidazole (MW 58.44g/mol) to the column 68.08g/mol), add about 900mL of deionized water, after stirring and dissolving, add NaOH to adjust the pH value of the solution to 8.0, add deionized water to make the volume to 1000mL) to wash the column, wash twice, 5ml each time.

(4) Elution: After washing, add Elution buffer (Elution buffer: NaH ₂ PO ₄ ·H ₂ O (MW 137.99g/mol) 6.9g, NaCl (MW 58.44g/mol) 17.54g, 17.00 g imidazole (MW 68.08g/mol), add about 900mL deionized water, stir to dissolve, add NaOH to adjust the pH value of the solution to 8.0, add deionized water to make up to 1000mL) to elute the protein, 400μl each time, wash 6 Second-rate.

(5) Collection: Different sterilized 1.5ml EP tubes were used for each elution for labeling and preservation.

(6) Sample processing: 10 μl of the eluted samples were drawn from each tube for SDS-PAGE identification ( FIG. 3 ).

(7) Preservation: According to the results of SDS-PAGE, add 20% (v/v) glycerol to the sample with higher purity of the target protein for preservation.

(8) BCA protein quantification: add the protein to 20% (v/v) glycerol for preservation, and use the BCA quantitative detection kit to detect its protein concentration (2.6695 mg/ml). The protein of the tested concentration was aliquoted and stored at -20°C.

Example 5 Detection of phage endolysin protein activity against Escherichia coli

Under the condition of pH=6, the activity of bacteriophage endolysin protein is detected, and the specific steps are as follows:

(1) The Escherichia coli strain BL21(DE3) was transferred to LB solid medium, and cultured at 37°C overnight.

(2) Pick part of the colonies in step (1) into LB liquid medium, and cultivate to OD600=1 on a shaker at 37°C and 200 rpm.

(3) Take 10 μl of the bacterial solution in step (2), use sterile PBS buffer (pH=6), and dilute it by 10 ⁴ times on a 96-well plate. That is, add 90 μl sterile PBS buffer (pH=6) to wells 1-4, add 10 μl of the bacterial solution in step (2) to the first well, repeat pipetting 5 times, and then add 10 μl of the first well liquid to the second well, Repeatedly pipetting 5 times, and so on until added to the 4th hole.

(4) Take 95 μl of the liquid from the fourth hole in step (3) and add it to the blank well, and add 5 μl of the protein purified and sterilized by filtration in Example 4 (for the blank group, add 5 μl of sterile PBS buffer with pH=6). liquid), mix by pipetting for 5 times, cover the lid, and incubate in a 37°C incubator for 16h.

(5) After the incubation, all the liquids in the blank group and the experimental group were taken out respectively, dropped on the LB medium, and the medium was evenly covered with the liquid by rotating the medium. After the liquid was dried, put it at 37°C for cultivation The box was inverted overnight, and the number of colonies was counted on the second day to determine whether the protein had antibacterial activity. The results were repeated three times. The results are shown in Figure 4.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

sequence listing

<110> South China Agricultural University

<120> A kind of anti-Escherichia coli phage endolysin and its application

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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

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Claims (10)

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;

(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.

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.

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