CN106565826B

CN106565826B - Escherichia coli O157: h7 affinity dodecapeptide, and screening method and application thereof

Info

Publication number: CN106565826B
Application number: CN201610979747.5A
Authority: CN
Inventors: 王平; 王宜冰; 施凡; 孙文; 甘凌风; 王超勇; 刘晓骏
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2016-11-08
Filing date: 2016-11-08
Publication date: 2020-03-20
Anticipated expiration: 2036-11-08
Also published as: CN106565826A

Abstract

The present invention discloses Escherichia coli O157: H7 affinity dodecapeptide and its screening method and application, and its amino acid sequence is shown in any one of SEQ ID NO.1 to SEQ ID NO.4. In the present invention, the food-borne pathogenic bacteria Escherichia coli O157:H7 is used as the target for screening, and several segments of polypeptides with strong affinity for the whole cell of Escherichia coli O157:H7 are obtained, and the negative screening step of discarding the centrifuge tube used for screening reduces the centrifugation The effect of the tube material polystyrene PS on the screening results; the screened phage monoclonal containing the affinity polypeptide has a stronger affinity for E. coli O157:H7 than the original peptide library; : The characteristics of H7 cell surface enrichment, the new lysozyme prepared by using Escherichia coli O157:H7 affinity dodecapeptide has the effect of targeting affinity to Escherichia coli, and has high enzymatic activity.

Description

Escherichia coli O157: h7 affinity dodecapeptide, and screening method and application thereof

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to Escherichia coli O157: h7 affinity dodecapeptide and its screening method and application.

Background

The incidence of food-borne diseases has increased over the past few years and has become a major public health problem worldwide. The food-borne pathogenic bacteria are an important cause for causing food-borne diseases, so that the analysis and detection of the food-borne pathogenic bacteria and the control of the spread of the food-borne pathogenic bacteria are of great significance. Escherichia coli O157: h7 is an important food-borne pathogenic bacterium, has wide harm and is easy to appear in foods such as vegetables, fruits and the like. And the cleaning and disinfection of such ready-to-eat food and the cleaning and disinfection of the instant food wherein escherichia coli O157: the detection of H7 has not been achieved to be rapid, safe, environmentally friendly and effective.

The screening of polypeptide by using phage display technology is an important research technology in the field of molecular biology. Panning of phage display peptide libraries can yield affinity peptides for materials or biomolecules as well as small organic molecules. There are many screens currently performed on materials, small molecules, as well as biomolecules and whole cells. And for pathogenic microorganisms escherichia coli O157: the affinity polypeptide of H7 has not been reported.

The antibacterial enzyme is used as a novel biological antibacterial agent, has the advantages of high efficiency, no toxicity, no residue and the like, and has wide prospect in the application of food industry, medical disinfection, detergents and the like. It faces some problems and challenges in practical application, mainly expressed in: the price of the enzyme is high; the action efficiency of the antibacterial enzyme is not high enough, so the usage amount is also increased; in practical applications, stability and environmental considerations also need to be taken into account. How to improve the enzyme activity and maintain the stability of the enzyme, and how to improve the targeting property and the utilization efficiency of the enzyme to target microorganisms are important concerns.

Lysozyme kills bacteria by hydrolyzing the cell wall peptidoglycan layer of certain bacterial species, and therefore it can be recommended as a natural antibacterial agent. However, lysozymes only act on gram-positive bacteria, so scientists are motivated to improve the antibacterial effect of lysozymes by several types of chemical modifications. In studies directed to the control effect of modified lysozyme on the growth of gram-positive and gram-negative bacteria in food products, the modification not only enhanced the functional properties of lysozyme (such as solubility and thermostability), but also extended the range of lysozyme activity. In general modified lysozyme is an excellent natural food detergent for use in the food industry.

Disclosure of Invention

The first object of the present invention is to provide Escherichia coli O157: h7 affinity dodecapeptide.

The second object of the present invention is to provide Escherichia coli O157: h7 method for screening affinity dodecapeptide.

The third object of the present invention is to provide Escherichia coli O157: h7 application of affinity dodecapeptide.

The fourth purpose of the invention is to provide a novel lysozyme.

The fifth purpose of the invention is to provide the application of the novel lysozyme.

In order to realize the first purpose of the invention, the invention discloses the following technical scheme: escherichia coli O157: h7 affinity dodecapeptide, which is characterized in that the amino acid sequence is shown in any one of SEQ ID NO. 1-SEQ ID NO. 4.

In order to realize the second purpose of the invention, the invention discloses the following technical scheme: escherichia coli O157: a method for screening H7 affinity dodecapeptide, which is characterized in that the screening method comprises the steps of screening Escherichia coli O157: h7 bacteria whole cell is used as target molecule, it is mixed with phage display dodecapeptide library, the peptide library is screened by centrifugal separation, and the influence of polystyrene PS material on the screening is removed by discarding the centrifugal tube used in the screening, finally the screened phage clone is DNA sequenced and analyzed, and ELISA is used to compare the affinity.

In order to achieve the third purpose of the invention, the invention discloses the following technical scheme: escherichia coli O157: application of H7 affinity dodecapeptide in preparing antibacterial material for colibacillus.

Escherichia coli O157: application of H7 affinity dodecapeptide in preparing detection reagent or detection chip for colibacillus.

Escherichia coli O157: the application of H7 affinity dodecapeptide in preparing novel lysozyme is characterized in that the dodecapeptide shown in any one of SEQ ID NO. 1-SEQ ID NO.4 is subjected to fusion expression with human lysozyme to obtain the novel lysozyme with affinity to escherichia coli.

Escherichia coli O157: the application of H7 affinity dodecapeptide in preparing food cleaning disinfectant.

In order to achieve the fourth object of the invention, the invention discloses the following technical scheme: a novel lysozyme is characterized in that the novel lysozyme is obtained by fusion expression of dodecapeptide shown in SEQ ID NO.1 and human lysozyme, and the amino acid sequence of the novel lysozyme is shown in SEQ ID NO. 5.

In order to achieve the fifth object of the invention, the invention discloses the following technical scheme: application of the novel lysozyme in preparation of a food cleaning disinfectant.

The amino acid sequences of the 4 dodecapeptides screened by the invention are respectively as follows: SEQ ID NO. 1: SGVYKVAYDWQH (P1); SEQ ID NO. 2: GLHTSATNLYLH (P2); SEQ ID NO. 3: VVSPDMNLLLTN (P3); SEQ ID NO. 4: VFSSMVHVLNTH (P4).

The amino acid sequence of the novel lysozyme obtained by the invention is SEQ ID NO. 5: MKALIVLGLVLLSVTVQGKVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACHLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGVGGGGSSGVYKVAYDWQH are provided.

The invention has the advantages that: the invention relates to a method for preparing a food-borne pathogenic bacterium escherichia coli O157: h7 is used as a target for screening, and the screening result is obtained according to the sequence shown in Escherichia coli O157: h7 polypeptide segments with strong whole cell affinity, and the negative screening step of discarding the centrifuge tube used for screening reduces the influence of polystyrene PS (polystyrene) material of the centrifuge tube on the screening result; the phage clones selected containing the affinity polypeptide were monoclonal to E.coli O157: h7 possesses stronger affinity than the original peptide library; the four screened polypeptides are obtained by screening in Escherichia coli O157: h7 cell surface enrichment characteristics. Provides a material basis and an effective way for developing antibacterial materials, food cleaning disinfectants, detection reagents or detection chips and the like based on the affinity polypeptide. Using Escherichia coli O157: the novel lysozyme prepared from the H7 affinity dodecapeptide has the effect of targeting affinity to escherichia coli, and has higher enzyme activity. The lysozyme has enzyme activity stronger than that of single lysozyme, has outstanding performance in the aspect of inhibiting the growth effect of pathogenic bacteria on an interface, is obviously better than the single lysozyme, has certain effect in the aspect of cleaning vegetables and fruits, and can be used as a food cleaning agent. This provides an effective way to develop a new generation of antibacterial agents and food cleaning disinfectants.

Drawings

FIG. 1 shows the frequency of amino acid residues in all the dodecapeptides obtained by screening.

FIG. 2 is a schematic diagram of the secondary structure of four polypeptide fragments.

FIG. 3 ELISA assay polypeptide P1 on E.coli O157: affinity of H7.

FIG. 4 is a histogram of the affinity of four fragments of polypeptide for three species of bacteria.

FIG. 5 shows the expression of four polypeptide fragments in E.coli O157: adsorption histogram on H7 mycoderm.

FIG. 6 Gene sequencing results.

FIG. 7 SDS-PAGE electrophoresis of expressed proteins, 1-5: before and after human lysozyme induction, culture solution, crushed supernatant, crushed sediment and 6: marker, 7-11, inducing by new lysozyme, culture solution, crushing supernatant and crushing precipitate.

FIG. 8 enzyme activity assay of expressed proteins.

FIG. 9 shows the bacteriostatic effect of glucose oxidase, lysozyme, catalase and fusion protein.

FIG. 10 comparison of the effect of lysozyme on the surface of vegetables and fruits with lysozyme containing affinity peptide.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1 enterobacter O157: h7 affinity dodecapeptide screening

The panning procedure is briefly described (reference: J.Phys.: condens. Matter 19(2007)395011(13 pp)):

1. selecting OD₆₆₀0.5O 157H 7 cells,add 10. mu.L of phage peptide library (10)¹¹) Suspended in 1ml PBS solution and incubated at room temperature for 60 min;

2. centrifuging at 16,000 Xg for 5min to precipitate bacteria and phage;

3. removing unadsorbed phage, washing with 1mL TBS/Tween buffer solution for 10 times, 16,000 Xg, 5min, and replacing new centrifuge tube for the last time;

4. the affinity phage and cells are finally eluted by 200 μ L of 0.2M glycine-HCl (pH2.2), gently shaken for 10min at room temperature, and centrifuged to take the supernatant;

5. finally, the mixture was neutralized with 150. mu.L of 1M Tris-HCl (pH 9.1).

6. Determining the phage titer:

① preparation of bacteria ER2738 was inoculated into 5mL LB medium and shaken at 37 ℃ to mid-log phase (OD600 about 0.5).

② agar plates were prepared by placing LB/IPTG/Xgal in a 37 ℃ incubator, prewarmed for use, one plate for each phage dilution gradient.

③ Top agar is prepared by melting top agar in a microwave oven, subpackaging into 3mL aliquots and sterilized test tubes, and incubating at 45 deg.C for use.

④ Serial gradient dilution of phage, eluting neutralized phage with LB medium for 10-fold gradient dilution, gradient range of 10¹-10⁴。

⑤ infection and plating, when the bacteria culture reaches the middle of the logarithm, the bacteria are divided into 200mL equal parts and put into a clean EP tube, each phage dilution gradient corresponds to one tube, 10 mul of phage with different dilutions are sucked and added into ER2738, the mixture is quickly oscillated and evenly mixed, the mixture is kept for 1min at room temperature, the infected cells are added into a top agar test tube with the temperature of 45 ℃, the mixture is one tube at a time, the mixture is quickly and evenly mixed and then immediately poured onto an LB/IPTG/Xgal plate with the temperature of 37 ℃, and the top agar is spread on the plate by gentle rotation.

⑥ cultivation and counting, after the plate is cooled for 5min at room temperature, it is cultivated overnight at 37 ℃ by inversion, and on the second day, a plate with about 100 plaques is selected for counting, and then the number is multiplied by the dilution factor of the phage of the plate to obtain pfu (plate formation units) per 10. mu.l of eluate.

7. Amplification of eluted phage

① A single clone of ER2738 was selected and inoculated into LB medium containing tetracycline for overnight culture, and diluted to 20mL of LB medium at a ratio of 1:100 the next day.

② the eluted and neutralized phages (about 80. mu.l) were added to 20mL of LB medium and cultured with vigorous shaking at 37 ℃ for 4.5 hours.

③ the culture was transferred to a 50mL centrifuge tube and centrifuged at 12,000g at 4 ℃ for 10min, and the supernatant was transferred to a new 50mL centrifuge tube and centrifuged again to remove residual cells and debris as much as possible.

④ collecting the supernatant about 80% to a new sterile centrifuge tube, adding 1/6 volume of 20% PEG/NaCl solution, mixing, and standing at 4 deg.C for at least 2 h.

⑤ 4 deg.C, 12000g centrifugal PEG precipitation for 15min, discard the supernatant and brief centrifugation, completely absorbing the residual supernatant, in the centrifugal tube with fingerprint size of phage precipitation residues.

⑥ the pellet was dissolved in 1mL TBS solution, the suspension was transferred to a 1.5mL sterile EP tube, centrifuged at 12,000rpm for 5min at 4 ℃ and the supernatant was transferred to a fresh sterile EP tube and mixed with 1/6 volumes of 20% PEG/NaCl solution and incubated on ice for 45 min.

⑦ 4 ℃, centrifugation at 14,000rpm for 10min, discarding the supernatant, centrifugation once again for a short time, sucking up the residual solution, then resuspending the pellet in 200. mu.l TBS solution, transferring the supernatant to a new sterile EP tube after centrifugation for 1min, and storing the amplified phage at 4 ℃ for titer determination and the next round of selection.

(7) The phage titer in the eluted amplificates was determined according to the method in step (5), and the phage titer obtained for each round of screening is shown in Table 1 below.

(8) And (3) carrying out a second round of screening: 2X 10 of the eluate amplified after the previous round of selection¹¹The phage amount of pfu repeats steps (2) - (4), adjusting the Tween concentration to 0.3% (v/v) in the washing step. Titers were determined after second round of eluate amplification for third round of screening.

(9) And (3) third screening: the second round of eluate was amplified and then titered for the third round of screening. A concentration of 0.4% (v/v) Tween20 was also used for the washing step. The fourth to fifth screening runs were performed as described above. The eluate obtained after the fifth round of screening was titered on LB/IPTG/Xgal plates. The plates can be stored in a 4 ℃ freezer and blue plaques selected for sequencing within 1-3 days.

(10) Amplification of plaques

① selecting ER2738 single clone to be inoculated into LB culture medium containing tetracycline for overnight culture, diluting the culture medium into LB culture medium according to the proportion of 1:100 the next day, and dividing 1mL of the culture medium into culture tubes for each tube, one culture tube is needed for each plaque, and blue plaques are selected into 1mL of bacteria-containing culture tubes on a plate for which the third round of eluate is measured.

Shaking culture at ② 37 deg.C for 4.5 h.

③ transferring the amplified bacterial liquid of the previous step to a 1mL clean EP tube, centrifuging for 30s, transferring the supernatant to another clean EP tube, centrifuging again, and transferring 80% of the supernatant to the clean EP tube.

(11) Plaque sequencing: the amplified plaques were subjected to DNA extraction using biomiga M13 single-stranded phage DNA extraction kit, and then sequenced by Shanghai Ruidi Biotech Ltd.

TABLE 1 phage titers obtained from each round of screening

Sequencing of monoclonal phages

1. Automatic sequencing was performed using-96 primers. The sequencing process is completed by Shanghai workers.

2. The sequencing results were analyzed by the methods described in the manual for phage peptide libraries to find the gene coding region for the random 7 peptide, and the corresponding amino acid sequences were written according to the manual providing a compact genetic code table (see table 2 below).

TABLE 2 simplified genetic code table

Elisa experimental protocol:

1. the host strain ER2738 was cultured overnight in a 5mL tube, and 5. mu.L tetracycline was added.

2. The overnight cultured ER2738 was diluted 1:100 in 20mL LB medium.

3. For each plaque clone to be identified, 5. mu.L of phage supernatant was added to each flask of ER2738 medium and incubated at 37 ℃ for 4.5hrs with aeration.

4. The above culture was transferred into a centrifuge tube and centrifuged at 10,000rpm for 10 min. The supernatant was transferred to a fresh centrifuge tube and centrifuged again.

5. 80% of the supernatant was placed in a fresh centrifuge tube and 1/6 volumes of PEG/NaCl were added. Precipitating at 4 deg.C for at least 1hr or overnight.

The pellet was centrifuged at 10,000rpm for 15min at 6.4 ℃ and the supernatant was discarded and centrifuged briefly again to remove the residual supernatant.

7. The pellet was resuspended in 1mL TBS and the suspension transferred to a microcentrifuge tube and centrifuged at 4 ℃ for 5min to remove residual cells from the pellet.

8. The supernatant was transferred to a fresh microcentrifuge tube and precipitated by adding 1/6 volumes of PEG/NaCl. Acting on ice for 15-60 min. Centrifuging at 4 deg.C for 10min, discarding supernatant, centrifuging for a short time, and removing residual supernatant by suction.

9. The pellet was resuspended in 50. mu.L TBS, 10. mu.L was taken for phage titer determination and incubated overnight at 37 ℃. The remaining phages were stored at 4 ℃.

10. The 96-well plates were coated with 100. mu.L of E.coli O157: H7 bacterial cells (dissolved in pH7.4PBS). Coating was carried out overnight at 4 ℃.

11. Excess target molecule solution is spun off, and the plate is inverted and patted on a paper towel to remove residual liquid. Each well was filled with blocking solution (skim milk in PBS/Tween). Before the phage is added to the target molecule coated plate, a microtiter plate is prepared for serial dilutions of phage, and the plate is blocked. Dilution in the other blocking plate alone is done to avoid phage adsorption to the target during dilution. All blocking plates were blocked for 1-2hrs at 4 ℃.

12. The blocking solution was spun off and the plate was washed 6 times with 1 × TBS/Tween, each time the plate was inverted and the wash solution was patted off on a clean paper towel, the Tween concentration should be the same as that used in the panning wash step (0.05%).

13. 200. mu.L TBS/Tween per well in separate blocking plates, 10 from the first well of each row¹²Starting from each virion, phage were diluted in a 2-fold gradient to well 12 at 4.8X 10⁸And (4) one virus seed.

14. Each line of diluted phage was added to the target molecule coated plate using a multichannel pipettor. Shaking at room temperature for 1-2 hrs.

15. The plate was washed 6 times with 1 × TBS/Tween (same as in step 12).

16. HRP-labeled anti-M13 antibody (GE) was diluted 1:5,000 in blocking solution. Add 200. mu.L of diluted antibody to each well and shake at room temperature for 1 hr.

17. The plate was washed 6 times with 1 × TBS/Tween (same as in step 12).

18. An HRP substrate solution was prepared as follows:

ABTS stock can be prepared in advance: 11mg of ABTS (Sigma # A1888) were dissolved in 50mL of 50mM sodium citrate solution (pH 4.0), filter sterilized and stored at 4 ℃. For each plate to be tested, 86 μ L of 30% H was added before the testing step₂O₂Add to 50mL of ABTS stock.

19. Adding 200 μ L substrate solution into each well, and reacting at room temperature for 10-60 min.

20. The absorbance at 405nm was recorded with a plate reader.

21. The affinity of the polypeptide P1 for E.coli O157: H7 is shown in FIG. 3.

Screening for polypeptide sequence Property analysis

Aiming at the single-stranded DNA sequence of the phage monoclone, the sequence alignment is carried out to obtain the DNA sequence inserted by the exogenous source, thereby deducing the amino acid sequence of the polypeptide, and the secondary structure of the polypeptide is obtained by a polypeptide calculator of the blaze organism (http://www.chinapeptides.com/toolcfuben.php) See fig. 1 and fig. 2.

A sequence table:

and (4) screening for multiple times to obtain 23 polypeptides in total, wherein the table above shows the occurrence probability of 7 polypeptides in 23 polypeptides. Wherein the repetition rate of P1-P4 is higher.

BCA kit method for measuring adsorption capacity of polypeptide for adsorbing three bacteria

1. Preparing working solution, namely preparing a proper amount of BCA working solution according to the number of the standard products and the samples and 50 volumes of BCA reagent A and 1 volume of BCA reagent B (50:1), and fully and uniformly mixing. The BCA working solution is stable within 24 hours at room temperature.

2. Diluting the standard substance: mu.l of the standard was diluted to 100. mu.l with PBS (the standard can be diluted with PBS in general) to give a final concentration of 0.5 mg/mL. The standard was added to the protein standard wells of a 96-well plate in 0,1,2,4,8,12,16, 20. mu.l, and PBS was added to make up to 20. mu.l.

3. Coli O157H 7, Bacillus subtilis and E.coli DH-5 α were cultured in 96-well plates

4. Appropriate volumes of polypeptide solution were added to the 96-well plates and supplemented with PBS to 20. mu.l.

5. 200. mu.l of BCA working solution was added to each well, and the mixture was left at 37 ℃ for 30 minutes.

6. Cooling to room temperature, and measuring A with microplate reader₅₆₂The protein concentration was calculated from the standard curve, from which the affinity of the four polypeptide fragments for the three bacteria was determined, as shown in FIG. 4.

The BCA kit method adsorbs four polypeptides to Escherichia coli O157: measurement of the amount of H7 biofilm adsorbed

3. Coli O157 in 96-well plates: h7 mycoderm.

6. Cooling to room temperature, and measuring A with microplate reader₅₆₂The protein concentration of the supernatant was calculated from the standard curve, from which four polypeptide fragments were determined for E.coli O157: affinity of H7, see fig. 5.

Example 2. use of E.coli O157: preparation of lysozyme from H7 affinity dodecapeptide

PCR primer design, synthesis and PCR amplification

Primer design and Synthesis

A forward primer:

5’—CTTTAAGAAGGAGATATACCATGGAT—3’(SEQ ID NO.9)

reverse primer:

5’—CCGCTCGAGTTAGTGCTGCCAGTCATAGGCCTCCTTATACACACCGCTGCTACCACCACCACCAACACCGCAAC—3’(SEQ ID NO.10)

PCR amplification of target genes

Screening the obtained polypeptide against E.coli:

p1: SGVYKVAYDWQH (SEQ ID NO.1), determined by codon preference of E.coli: AGCGGUGUGUAUAAGGUGGCCUAUGACUGGCAGCAC (SEQ ID NO.13)

And (3) PCR reaction system: primer, polymerase, template (human lysozyme hly) and buffer solution

PCR reaction thermal cycling program: performing pre-denaturation at 95 ℃ for 5min, and then performing circulation; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and 30 cycles; finally, extension is carried out for 10min at 72 ℃.

Construction of cloning vectors

Ligation of target Gene to vector

Polypeptide P1 obtained by screening in a laboratory and vector plasmid pET28a are synthesized by segmentation, a sequence for coding a kinase recognition site and a Thrombin enzyme cutting site are introduced into the upstream 5 'end of P1 by a PCR method, and a stop codon and a BamH I enzyme cutting site are introduced into the downstream 5' end.

The positive cloning plasmid is double digested by Nde I/BamH I, the DNA fragment coding for P1 is recovered and connected with pETl5b which is double digested in the same way, Ecoli DH5 α competent cells are transformed, the positive clone is screened, and PCR and restriction enzyme digestion identification are carried out.

Finally obtaining the DNA fragment coding the P1-lysozyme fusion protein by a PCR bridging method. The target protein of the fragment is cloned into a prokaryotic expression vector pET28a, so as to obtain a recombinant plasmid. With the forward primer:

5’—CTTTAAGAAGGAGATATACCATGGAT—3’(SEQ ID NO.11)

reverse primer:

5’—CCGCTCGAGTTAGTGCTGCCAGTCATAGGCCTCCTTATACACACCGCTGCTACCACCACCACCAACACCGCAAC—3’(SEQ ID NO.12)

a DNA fragment encoding the P1-lysozyme fusion protein was amplified for the upstream and downstream primers and ligated into E.coli expression vector pET-28a to obtain a recombinant expression plasmid. And a control recombinant expression plasmid was obtained according to the above-mentioned method. The plasmid was transformed into E.coli, and kanamycin-resistant recombinant transformants were selected and sequenced by Shanghai Ruidi Biotech Ltd, the sequencing results of which are shown in FIG. 6.

Induced expression and renaturation of target gene

Induced expression of a Gene of interest

From the 4C and 5C cultures, 1mL of each culture was added to the Erlenmeyer flask culture medium and incubated for 2h at 37 ℃ on a shaker.

50. mu.L of each of the bacterial solutions was inoculated into a Erlenmeyer flask medium and cultured overnight in a shaker at 37 ℃.

Extraction of the expression product

The whole culture was placed in a centrifuge tube, centrifuged (8000rpm) and centrifuged for two minutes. The supernatant was decanted off and the remaining bacterial pellet was shaken up with PB7.4 buffer.

The bacterial pellet is broken by a vacuum cell breaker, and the protein is broken (repeating the operation 2-3 times), placed in a cup filled with ice, and then stored in a refrigerator until the next operation step.

Protein detachment:

the separation column was filtered twice with PB solution, the bacterial lysate was poured in, and after filtration, a needle washed with PB7.4 buffer solution was attached. 10mL of 50mmol/L pyrazole solution is poured, and the liquid flowing down is connected with a centrifuge tube; adding 10mL of 100mmol/L pyrazole solution and then flowing down the liquid; 10mL of a 200mmol/L pyrazole solution was added and the liquid was poured. Removing the needle, adding about 10mL of 500mmol/L pyrazole solution, filtering, adding 70% alcohol, filtering, sealing the outlet when half of alcohol is left, and storing for later use. The 4C and 5C bacterial crushing liquid is operated once according to the steps, and the obtained separation product is stored in a refrigerator.

Renaturation and gradient renaturation of Inclusion bodies

And (3) dialysis renaturation:

pretreatment of the dialysis bag:

at 2% NaHCO₃And 1mM EDTA (pH 8.8) for 10min, thoroughly washing the dialysis bag with ultrapure water, boiling in 1mM EDTA (pH 8.8) for 10min, cooling, and storing in 20% ethanol.

Washing of inclusion bodies:

the washing solution was added in a ratio of 100mL of the cell disruption solution to 10mL of the washing solution. In this experiment, 200mL of each of the 4C and 5C tubes was added with 20mL of each wash solution. Each wash was 10min and centrifuged for 15min (1 mL of supernatant and 2. mu.g of pellet were retained). The remaining pellet was washed twice more, and 1mL of supernatant and 2. mu.g of pellet were retained after each centrifugation.

Preparing a washing solution: 15% glucose, pH8.0, 5mmol/L EDTA, 0.5mmol/L PMSF, 1% Triton x-100 PBS.

Dissolving the inclusion body:

and (3) crushing thalli: adding the solution at a ratio of 100:10, stirring for 2h, centrifuging the inclusion body solution at 4 deg.C and 10000rpm for 15min (1 mL of precipitate and supernatant are respectively sampled, and the precipitate is dissolved with PBS), collecting supernatant, and dialyzing.

Preparation of a lysis solution (protein-renaturation lysis solution): 50mmol/L Tris-HCl,5mol/L guanidine hydrochloride, pH 8.0.

And (3) dialysis:

putting the supernatant protein into a dialysis bag, and clamping two sides of the dialysis bag by a special clamp to prevent the protein liquid from leaking. The bag containing the protein solution was then placed in dialysate (500mL) and dialysis was performed for 6 hours. Dialysis was repeated twice. All of the above steps were performed in an ice bath or at 4 ℃ to ensure that the fusion protein activity was not lost.

Preparing a dialysate: 50mmol/L Tris-HCl, 0.1% Triton x-100, pH 8.0.

Preparing a Tris-HCl solution: 0.5mol/L Tris pH8.0.

Gradient renaturation:

preparing a dialysate:

(1)4mol/L guanidine hydrochloride, 50mmol/L Tris-HCl, 0.1% Triton x-100, pH 8.0.

(2)2mol/L guanidine hydrochloride, 50mmol/L Tris-HCl, 0.1% Triton x-100, pH 8.0.

(3)1mol/L guanidine hydrochloride, 50mmol/L Tris-HCl, 0.1% Triton x-100, pH 8.0.

(4)0mol/L guanidine hydrochloride, 50mmol/L Tris-HCl, 0.1% Triton x-100, pH 8.0.

Putting the supernatant protein (5C) into a dialysis bag, and clamping two sides of the dialysis bag by using a special clamp to prevent the leakage of protein liquid. The bag containing the protein solution was then placed in dialysate (500mL) and dialysis was performed for 6 hours. Dialyzing from high to low according to the above dialysis concentration for 6 hours each time for four times.

The electrophoresis of the expressed protein is shown in FIG. 7.

Enzyme activity assay for lysozyme

Preparation of a culture medium: 5g of peptone; 3g of beef extract; 5g of sodium chloride; 20g of agar powder; 1000mL of distilled water; adjusting the pH value to 7.0-7.2.

Preparation of bacterial suspension: inoculating Micrococcus Lysodeikcicus (Micrococcus lysodeikcicus) to a liquid culture medium, wherein the temperature of a shaking table is 35 ℃, the rotating speed is 200r/min, and the culture time is 24 h; and (3) putting the liquid after the enlarged culture into a centrifuge tube, centrifuging (4000r/min, 20min), discarding supernatant, obtaining precipitate, namely the thallus, adding a small amount of 0.9% sterilized normal saline to wash the thallus, centrifuging again, discarding supernatant, repeatedly washing the centrifuged thallus for 3 times, adding a small amount of 20% sterilized glycerol as a protective agent into the obtained thallus precipitate, and freezing and storing in a refrigerator. Thawing in ice bath, grinding in a sterilized mortar for 20min, and diluting the ground suspension with phosphate buffer solution of pH 6.2Measuring OD value at 450nm, and regulating OD of bacterial suspension₄₅₀Around 1.0.

And (3) activity determination: respectively placing the enzyme solution and the bacterial suspension to be detected in a water bath at 25 ℃ for 30min, sucking 2.8mL of the bacterial suspension, placing the bacterial suspension in a 1cm cuvette, measuring the OD value at 450nm, reading when the OD value is zero, then adding 0.2mL of the enzyme solution, quickly shaking up, timing after adding the enzyme solution, and measuring the OD once every minute₄₅₀And the total number of measurements was three. The unit of activity of the enzyme in this test is defined as the decrease of 0.001 of OD value per minute as one unit of activity (25 ℃, pH 6.2).

Enzyme activity (U) ═ Δ OD₄₅₀/min)*10³

Specific enzyme activity (U/mg) ═ Δ OD₄₅₀/min)*10³Mg/sample

The results are shown in FIG. 8.

Verification of antibacterial Activity in bulk solution, at interfaces

96-well plate method:

escherichia coli was inoculated in a test tube medium and cultured overnight in a shaker at 37 ℃.

Each well of the plate: adding 100 mul of Escherichia coli bacterial liquid, adding 50 holes, and culturing in shaker at 37 deg.C for more than 12 hr.

Each well of the plate: sucking out the culture medium, adding 200 μ l PBS buffer solution, washing for 3 times, adding 15 μ l methanol, and naturally drying at room temperature for 15 min.

Select 3-5 wells: add 100. mu.l of 0.1% crystal violet stain to stain for 5min, aspirate the stain and wash with PBS buffer.

The observation of the presence or absence of the adsorption of Escherichia coli under a microscope showed that no Escherichia coli was observed, so that Escherichia coli was not adsorbed on the plate by forming a film, and could be verified only by a simple method (plate drawing).

A flat plate scribing method:

mu.l of lysozyme was added to 100. mu.l of E.coli, and streaking was performed to compare the number and size of colonies, thereby comparing the antibacterial effect of each antibacterial enzyme on E.coli. 2mg of each enzyme was dissolved in 1mL of sterile PBS buffer solution to prepare an enzyme solution with a concentration of 2mg/mL, or 2. mu.l of each enzyme was dissolved in 1mL of sterile PBS buffer solution to prepare an enzyme solution with a concentration of 2. mu.l/mL, and the enzyme solution was placed in a refrigerator for further use. Taking 4 centrifugal tubes, respectively adding 100 mu l of Escherichia coli O157 bacterial liquid, and then respectively adding 100 mu l of enzyme solution. Then, an inoculating loop is used for quickly dipping escherichia coli O157 bacterial liquid for plate streaking. The results are shown in FIG. 9.

Note that the sample handling steps are consistent each time, and the time duration is controlled within a consistent range. All steps are operated under the condition of a sterile super clean bench so as to ensure that no other mixed bacteria are mixed, thereby influencing the correctness and the comparability of experimental results.

Example 3 actual removal of bacteria from the surface of vegetables and fruits

The fruit used in the experiment is a small tomato, and the comparison shows that the lysozyme antibacterial effect in various commercial antibacterial enzymes is better, so the comparison of the actual vegetable and fruit surface bacteria cleaning effect is mainly carried out by using the lysozyme and the fusion protein. The operation steps, time and conditions of each sample are controlled under the same condition to eliminate the influence of other interference factors on the experimental result, thereby ensuring the accuracy and reliability of the experimental result of the comparison of the antibacterial effect of the lysozyme and the fusion protein.

In water at a ratio of 50: 2, fusion protein, lysozyme and blank were added to compare the cleaning effect (fruit cleaning effect was compared by plating and plate streaking).

Adding 2mL of expression product fusion protein into 50mL of ultrapure water to clean three small tomatoes; adding 2mL of 2mg/mL of lysozyme into 50mL of ultrapure water to wash three small tomatoes; three small tomatoes were washed with 50mL of ultrapure water. After the three are cleaned, streak culture and plate coating culture comparison are carried out. The results are shown in FIG. 10.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> university of east China's college of science

<120> Escherichia coli O157: h7 affinity dodecapeptide, and screening method and application thereof

<130>/

<160>13

<170>PatentIn version 3.5

<210>1

<211>12

<212>PRT

<213> Artificial Synthesis

<400>1

Ser Gly Val Tyr Lys Val Ala Tyr Asp Trp Gln His

1 5 10

<210>2

<211>12

<212>PRT

<213> Artificial Synthesis

<400>2

Gly Leu His Thr Ser Ala Thr Asn Leu Tyr Leu His

1 5 10

<210>3

<211>12

<212>PRT

<213> Artificial Synthesis

<400>3

Val Val Ser Pro Asp Met Asn Leu Leu Leu Thr Asn

1 5 10

<210>4

<211>12

<212>PRT

<213> Artificial Synthesis

<400>4

Val Phe Ser Ser Met Val His Val Leu Asn Thr His

1 5 10

<210>5

<211>165

<212>PRT

<213> Artificial Synthesis

<400>5

Met Lys Ala Leu Ile Val Leu Gly Leu Val Leu Leu Ser Val Thr Val

1 5 10 15

Gln Gly Lys Val Phe Glu Arg Cys Glu Leu Ala Arg Thr Leu Lys Arg

20 25 30

Leu Gly Met Asp Gly Tyr Arg Gly Ile Ser Leu Ala Asn Trp Met Cys

35 40 45

Leu Ala Lys Trp Glu Ser Gly Tyr Asn Thr Arg Ala Thr Asn Tyr Asn

50 55 60

Ala Gly Asp ArgSer Thr Asp Tyr Gly Ile Phe Gln Ile Asn Ser Arg

65 70 75 80

Tyr Trp Cys Asn Asp Gly Lys Thr Pro Gly Ala Val Asn Ala Cys His

85 90 95

Leu Ser Cys Ser Ala Leu Leu Gln Asp Asn Ile Ala Asp Ala Val Ala

100 105 110

Cys Ala Lys Arg Val Val Arg Asp Pro Gln Gly Ile Arg Ala Trp Val

115 120 125

Ala Trp Arg Asn Arg Cys Gln Asn Arg Asp Val Arg Gln Tyr Val Gln

130 135 140

Gly Cys Gly Val Gly Gly Gly Gly Ser Ser Gly Val Tyr Lys Val Ala

145 150 155 160

Tyr Asp Trp Gln His

165

<210>6

<211>12

<212>PRT

<213> Artificial Synthesis

<400>6

Gly Ser Ala Pro Leu Leu Thr Val Asp Thr Ser Lys

1 5 10

<210>7

<211>12

<212>PRT

<213> Artificial Synthesis

<400>7

Cys Tyr Ala Gly His Asp Leu Tyr Val Ala Ala Asp

1 5 10

<210>8

<211>12

<212>PRT

<213> Artificial Synthesis

<400>8

Ser Leu Ser Trp Leu Thr Lys Met Gln Met Glu Met

1 5 10

<210>9

<211>26

<212>DNA

<213> Artificial Synthesis

<400>9

ctttaagaag gagatatacc atggat 26

<210>10

<211>74

<212>DNA

<213> Artificial Synthesis

<400>10

ccgctcgagt tagtgctgcc agtcataggc ctccttatac acaccgctgc taccaccacc 60

accaacaccg caac 74

<210>11

<211>26

<212>DNA

<213> Artificial Synthesis

<400>11

ctttaagaag gagatatacc atggat 26

<210>12

<211>74

<212>DNA

<213> Artificial Synthesis

<400>12

ccgctcgagt tagtgctgcc agtcataggc ctccttatac acaccgctgc taccaccacc 60

accaacaccg caac 74

<210>13

<211>36

<212>RNA

<213> Artificial Synthesis

<400>13

agcggugugu auaagguggc cuaugacugg cagcac 36

Claims

1. Escherichia coli O157: h7 affinity dodecapeptide, characterized in that, the amino acid sequence is shown in SEQ ID NO. 4.

2. Escherichia coli O157: h7 affinity dodecapeptide in preparation of polypeptide for Escherichia coli O157: the application of the antibacterial material H7 is characterized in that the amino acid sequence of the affinity dodecapeptide is shown in any one of SEQ ID NO. 1-SEQ ID NO. 4.

3. Escherichia coli O157: h7 affinity dodecapeptide in preparation of polypeptide for Escherichia coli O157: the application of the detection reagent or the detection chip of H7 is characterized in that the amino acid sequence of the affinity dodecapeptide is shown in any one of SEQ ID NO. 1-SEQ ID NO. 4.

4. Escherichia coli O157: the application of H7 affinity dodecapeptide in preparing lysozyme is characterized in that fusion expression is carried out on dodecapeptide shown in any one of SEQ ID NO. 1-SEQ ID NO.4 and human lysozyme to obtain the recombinant human lysozyme for Escherichia coli O157: h7 lysozyme with affinity.

5. A lysozyme is characterized in that the dodecapeptide shown in SEQ ID NO.1 and human lysozyme are subjected to fusion expression to obtain the lysozyme, and the amino acid sequence of the lysozyme is shown in SEQ ID NO. 5.

6. Use of lysozyme according to claim 5 for the preparation of a food cleaning disinfectant.