CN109206484B

CN109206484B - Peptide segment for preventing and treating enteropathogenic escherichia coli infection

Info

Publication number: CN109206484B
Application number: CN201810651430.8A
Authority: CN
Inventors: 邓启文; 余治健; 陈重; 程航; 邓向斌; 李多云; 郑金鑫
Original assignee: SHENZHEN NANSHAN DISTRICT PEOPLE'S HOSPITAL
Current assignee: SHENZHEN NANSHAN DISTRICT PEOPLE'S HOSPITAL
Priority date: 2015-11-20
Filing date: 2015-11-20
Publication date: 2021-06-18
Anticipated expiration: 2035-11-20
Also published as: CN109180786B; CN109180786A; CN109206484A; CN108976289B; CN108976289A; CN105294840A; CN105294840B

Abstract

The invention provides a peptide fragment for preventing and treating enteropathogenic escherichia coli infection, which comprises the amino acid sequence shown in SEQ ID NO: 2. By adopting the technical scheme of the invention, the affinity between the polypeptide containing the peptide segment for preventing and treating enteropathogenic escherichia coli infection and the EspB protein is higher than that of the control protein, and the polypeptide capable of preventing and treating enteropathogenic escherichia coli infection can inhibit the adhesion of EPEC and HEp-2 cells and block the function of EspB, thereby reducing the adsorption of EPEC on epithelial cells, providing a new direction for EPEC treatment and laying a foundation for developing medicines for preventing and treating EPEC.

Description

Peptide segment for preventing and treating enteropathogenic escherichia coli infection

The application is application number: 201510811930X, filing date: 11/20/2015, patent name: a divisional application of a polypeptide for the prevention and treatment of enteropathogenic E.coli infection.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a peptide fragment for preventing and treating enteropathogenic escherichia coli infection.

Background

At present, the enteropathogenic escherichia coli is treated by broad-spectrum antibiotics, and no inhibitor is specifically used for the bacteria. The antibiotic therapy is easy to generate drug resistance, so that drug-resistant variant bacteria are gathered in a human body, and the subsequent infection therapy is difficult.

Enteropathogenic escherichia coli (EPEC) is one of the important pathogenic bacteria causing diarrhea in infants and young children worldwide and sporadic diarrhea in adults, and current studies indicate that EPEC causes damage to the intestinal mucosa mainly through adhesion and exfoliation injury (a/E injure). The enteropathogenic Escherichia coli EPEC is characterized in that pathogenic bacteria can naturally adhere to host cell membranes, destroy cell microvilli, and induce the formation of a cup-like basement membrane by cytoskeletal protein under the adhesion of bacteria, which is called as adhesion and shedding damage. The characteristic of the EPEC in the effects of adhesion and shedding is that the EspB, EspD and EspA transporters are relied on to form close adhesion to host cells, and the adhesion cells remove microvilli and accumulate fiber actin. The EspB protein can interact with the EspD protein to be inserted into a host cell membrane to form micropores, so that EPEC virulence factors can directly enter the host cell through the micropores formed on the cell membrane, and the invaded virulence factors promote the formation of bacterial adhesion and floating effects. The EspB-deleted mutant EPEC cannot mediate extension of microvilli adjacent to adherent cells, nor prevent phagocytosis by macrophages. Because the EspB protein is a key protein for enteropathogenic Escherichia coli, if a substance capable of inhibiting the pathogenic effect of EPEC can be found, a new strategy is searched for EPEC treatment, and the EPEC can be prevented and treated in the future.

Disclosure of Invention

Aiming at the technical problems, the invention discloses a peptide segment for preventing and treating enteropathogenic escherichia coli infection, which is proved to be capable of inhibiting the pathogenic effect of EPEC, blocking the pathogenicity of EPEC in a targeting manner and laying a foundation for developing a medicament for preventing and treating EPEC.

In contrast, the technical scheme adopted by the invention is as follows:

a peptide stretch for use in the prevention and treatment of enteropathogenic e.coli infection comprising the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4.

The invention also provides nucleic acid molecules encoding polypeptides comprising a peptide stretch as described above.

The invention also provides an expression cassette, a recombinant vector, a transgenic cell line or a recombinant bacterium containing the nucleic acid molecule.

The invention also provides a composition containing the polypeptide of the peptide segment for preventing and treating enteropathogenic escherichia coli infection.

Further preferably, the composition further comprises a bactericide.

Further preferably, the composition further comprises a non-polypeptide ingredient added to achieve a function and effect other than bactericidal.

The invention also provides the use of a polypeptide as described above, a nucleic acid molecule as described above or a composition as described above for the manufacture of a medicament for killing or inhibiting enteropathogenic escherichia coli.

Compared with the prior art, the invention has the beneficial effects that:

by adopting the technical scheme of the invention, the affinity between the polypeptide containing the peptide segment for preventing and treating enteropathogenic escherichia coli infection and the EspB protein is higher than that of a control protein, and in vitro research shows that the polypeptide for preventing and treating enteropathogenic escherichia coli infection can inhibit the adhesion of EPEC and HEp-2 cells and block the function of EspB, thereby reducing the adsorption of EPEC on epithelial cells, providing a new direction for EPEC treatment and laying a foundation for developing a medicament for preventing and treating EPEC.

Drawings

FIG. 1 is a Western Blot method for detecting EspB protein expression. In the figure, A is an expression condition diagram after a constructed pET21b-EspB expression plasmid is transfected into Escherichia coli, a mouse anti-human 6-histidine monoclonal antibody is a main antibody, 1 is a bacterial lysate of EspB expression vector transfection, 2 is a culture supernatant expression recombinant EspB protein, and 1 and 2 respectively show the bacterial lysate of EspB expression vector transfection and the condition that the culture supernatant expression recombinant EspB protein. B is a purified EspB protein expression diagram.

FIG. 2 is an identification chart of 12 positive phages screened out in the invention, a control of vcSM13, EspB and 6-histidine short peptide by an ELISA method for affinity, each experiment is repeated for more than 3 times, and the average number is taken as final analysis data.

Detailed Description

The technical scheme of the invention is further explained in detail with reference to the attached drawings.

1. Expressing EspB protein in vitro;

the EspB fragment was amplified from E2348/69 (O127: H6) strain and inserted into the BamHI and EcoRI sites of the pET21b expression vector using standard molecular methods to construct a pET21b-EspB expression vector containing 6 (His) histidine residues. The recombinant E.coli transfected with pET21b-EspB expression vector was induced to express the EspB protein by adding IPTG to the medium. Cells were collected by centrifugation after addition of 1mM IPTG and suspended using Qiagen buffer A, 20mM Tris,500mM NaCl, pH 9 from buffer A. Centrifuging the cell suspension after cracking the cells by a sonic cracking method, and collecting the supernatant for Ni²⁺Exchange membrane (Qiagen Corp.) filtration. The EspB protein containing the His tag was eluted with an eluent (Qiagen), and the protein was analyzed and collected by Western blot using a mouse anti-His monoclonal antibody. The concentration of recombinant His-EspB protein in the solution was quantitatively collected by the Bradford method (Bio-Rad, Hercules, Calif., USA) and then stored in a refrigerator at-70 ℃.

Screening of EspB specific binding proteins

The EspB specific binding protein was selected using phage M13 using a 12-mer random library (New England BioLabs, Ipswich, MA, USA). 150 μ L of a solution containing 10 μ g/mL of EspB protein and 0.1M sodium bicarbonate was added to a sterile polystyrene petri dish and shaken repeatedly until the surface of the dish was completely wet. The ESpB protein coated dishes were deblocked with calf protein serum and washed with 0.05% PBST buffer. 10 μ L of stock solution diluted with PBST was added to the medium and incubated at room temperature for 2 h. After washing the coated dishes with 0.05% PBST, the remaining adherent phage were eluted for amplification. After three rounds of amplification, the eluate was amplified in ER2738 medium, purified by polyethylene glycol precipitation and quantified according to the instructions for the use of the reagents.

Screening of Positive phage by ELISA method

Coating 96-well plate with EspB protein and control 6-histidine peptide overnight, blocking buffer, adding 10 per well¹⁴The phage clone or the control sample of pfu is incubated for 1h at normal temperature; adding horseradish peroxidase labeled M13, and incubating for 1h at normal temperature; adding 50 mu L/hole of benzidine; after 20min, 50. mu.L/well of 2M H was added₂SO₄Terminating the reaction; and detecting the absorbance (A) value of the sample at the wavelength of 450 nm. If the A value of the sample is more than 2 times higher than the control phage and peptide, the target protein is considered to have higher affinity with the EspB protein.

DNA sequencing and peptide chain Synthesis

Amplifying DNA fragments in positive phage according to a 12-mer random library of New England BioLabs, Ipswich, MA, USA reagent operating instruction, and sending to sanger for sequencing; the DNA sequence was then analyzed using the BioEdit sequence alignment editing software. The peptide chain of interest was synthesized by Sigma-Aldrich and was greater than 98% pure. A short C addition of 6 histidines to the Gly-Gly-Gly-Ser spatial structure served as a negative control.

5. Cell viability assay

HEp-2 cells at 2X 10³The culture medium is inoculated to a 96-well culture plate at a density of one well, the culture medium is changed into a DMEM medium containing 1% calf serum to be cultured for 24 hours after the culture medium is adhered to the wall overnight, and the culture medium is further incubated for 48 hours by a series of short peptides diluted in a gradient manner. Cell viability was analyzed by MTT staining, and absorbance at 490 nm represents the relative viable cell number.

HEp-2 cell inhibition assay

Reference to Infect immun.2006; 74(12) 6920-8; j Agric Food chem.2013; 61(11) 2748-54.JFood prot.2008; 71(11) 2272-7 the method described therein uses the EPEC E2348/69 strain for inhibition assays. Before the assay, the EPEC strain was incubated with 4ml of a sugarless trypsin medium, followed by washing with 5% calf serum in DMEM and 1% SDMEM.EPEC cells grown on a slant petri dish were inoculated into fresh 1% SDMEM medium and locally adsorbed. HEp-2 cells were suspended in Gibco medium containing 10% calf serum at 5X 10⁴Cell density per well was seeded into 24-well culture plates. To every 10⁷Adding a series of short peptides with the concentration of 10,50,100 and 200 mu g/mL into the cells; the negative control group was added with 6 histidine oligopeptide at a concentration of 200. mu.g/mL. Plates were incubated at 37 ℃ CO₂Incubate for 30 min, after which time the nonadherent cells are removed by washing with PBS buffer. The petri dish was fixed with methanol for 10 minutes, dried, stained by Giemsa method for 20 minutes, and then observed under a contrast microscope with a magnification of 100 ×. At least 100 consecutive HEp-2 s can be observed at this magnification. If one cell comprises 4 or more than 4 EPEC bacteria, the cell is considered as a local adsorption positive cell and has the phenotypic characteristic; the ratio of locally adsorbed cells per 100 consecutive cells represents the adsorption rate. Each experiment was repeated three times or more, and the reduction ratio was calculated using the following formula.

The reduction ratio is (average of negative control group-average of experimental group)/positive control group.

7. Statistical analysis

The study data was analyzed using software GraphPad Prism v 5.01. The difference between the two groups was examined by T. The comparisons between groups were performed using one-way analysis of variance. P <0.05 was considered significantly different.

8. Results of the experiment

As shown in figure 1, the constructed pET21b-EspB expression plasmid is transfected into Escherichia coli to successfully express the EspB protein, and a Western blot method is used for verifying protein expression and purifying the protein, the verification result is shown in figure 2, as can be seen from figure 2, a bright band is formed at 37KD, and the EspB protein is highly expressed.

Enrichment experiments were performed on EspB protein-binding phages and the results are detailed in table 1. As can be seen from Table 1, the Ph.D.12 methionine phage display technology is used for screening the EspB specific binding protein, and three rounds of affinity selection test results show that all the phages capable of specifically binding the EspB protein are enriched, and 12 clones screened preliminarily possibly have a binding effect with the EspB protein.

TABLE 1 enrichment of EspB protein binding to phages

The affinity of 12 selected positive phages and the control of vcsM13 with EspB and 6-histidine short peptide was identified by ELISA, each experiment was repeated 3 times more, the average was taken as the final analytical data, the results are shown in fig. 2, and as can be seen from fig. 2, each phage clone was incubated with EspB protein alone.

And analyzing and screening the phages, namely peptide-6, peptide-7, peptide-8 and peptide-12, with high affinity to the EspB protein by adopting an ELISA method. According to the sequencing result of sanger, the four peptide sequences are respectively as follows:

peptide-6: YFPYSHTSPRQP; (as shown in SEQ ID NO: 1)

peptide-7: AYKYT SALPAEA; (as shown in SEQ ID NO: 2)

peptide-8: SLTLMNSPLGAS; (as shown in SEQ ID NO: 3)

peptide-12: MLTLSLNPTNSA; (as shown in SEQ ID NO: 4)

Peptide-6, peptide-7, peptide-8 and peptide-12 were subjected to MTT assay, and the data of MTT assay are shown in Table 2. As can be seen from Table 2, peptide-6, peptide-7, peptide-8 and peptide-12 have no obvious toxicity to HEp-2 cells, and the research results show that peptide-6 can significantly reduce the adsorption ratio of EPEC to HEp-2 cells with the increase of the concentration, and when the concentration is 100 mug/mL, the adsorption ratio of EPEC to HEp-2 cells is reduced by 40% compared with the control group.

TABLE 2 Effect of polypeptides on EPEC-adherent HEp-2 cells in vitro

By adopting the technical scheme of the embodiment, a phage display technology is adopted to search the polypeptide specifically bound to the EspB protein, 12 short peptides are screened in 3 rounds of enrichment experiments, and an ELISA method is adopted to identify 4 strips which have better affinity with the EspB and can be used for treating EPEC infection.

EspA, EspB and EspD proteins secreted by EPEC can form a needle-tip-like micropore structure on a host cell membrane, and we can select EspB specific binding protein, and block the formation of needle-tip-like micropores by binding the EspB protein, thereby inhibiting the adsorption of EPEC on host cells.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.

Claims

1. A peptide fragment for use in the prevention and treatment of enteropathogenic e.coli infection, characterized by: it is SEQ ID NO: 2.

2. Nucleic acid molecule encoding a peptide stretch for use in the prevention and treatment of enteropathogenic E.coli infection according to claim 1.

3. An expression cassette, recombinant vector, transgenic cell line or recombinant bacterium comprising the nucleic acid molecule of claim 2.

4. A composition comprising the polypeptide of claim 1 for the prevention and treatment of enteropathogenic e.

5. The composition of claim 4, wherein: also comprises a bactericide.

6. The composition of claim 4, wherein: also comprises non-polypeptide components added for realizing functions and effects except for sterilization.

7. Use of the peptide fragment for the prevention and treatment of enteropathogenic e.coli infection according to claim 1 for the preparation of a medicament for inhibiting enteropathogenic e.coli.

8. Use of the nucleic acid molecule of claim 2 in the manufacture of a medicament for inhibiting enteropathogenic large stalk bacteria.

9. Use of a composition according to claim 4, 5 or 6 for the preparation of a medicament for inhibiting enteropathogenic large stalk bacteria.