CN110714000A - Application of Toll-like receptor ligand protein in resisting bacterial infection - Google Patents

Abstract

The invention provides an application of Toll-like receptor ligand protein in resisting bacterial infection. The streptococcus pneumoniae endopeptidase O (PepO) is a ligand of a Toll-like receptor 2 and a Toll-like receptor 4, can remarkably enhance the phagocytosis and the killing effect of macrophages on pathogenic bacteria, and can up-regulate the secretion of related cytokines and chemokines, thereby inducing strong innate immune response and enhancing the resistance of respiratory tracts to pathogenic bacteria infection.

Description

Application of Toll-like receptor ligand protein in resisting bacterial infection

Technical Field

The invention relates to the technical field of medical biology, in particular to application of Toll-like receptor ligand protein in resisting bacterial infection, and especially relates to application of streptococcus pneumoniae endopeptidase O in resisting bacterial infection.

Background

Antibiotics are main drugs for treating bacterial infectious diseases, however, due to extensive and long-term abuse of antibiotics, explosive emergence of drug-resistant bacteria and even multiple drug-resistant bacteria is promoted, and the clinical curative effect of the antibiotics is increasingly poor. Among the twelve major Health threats listed in the World Health Organization (WHO) worldwide in 2019, one is antibiotic resistance. Therefore, bacterial infection is still one of the main problems threatening human health at present, and the development of new therapeutic drugs is urgently needed. However, the development of new antibiotics is far from meeting clinical needs, and therefore, there is a great need to find new strategies to cope with bacterial infectious diseases.

It is well known that there are many immune defense mechanisms against pathogens, and the occurrence of pathogen infection is associated with the weakened or disturbed immunity of the body, so that it should be an effective means for resisting pathogens by regulating the body's own immune function. The research idea has been successful in the immunotherapy of tumors, and the representative result is the development and application of immune checkpoint inhibitors (such as inhibitors targeting PD-1/PD-L1 and CTLA-4).

Toll-like receptors (TLRs) are important pattern recognition receptors, and participate in a series of immune reactions after recognizing pathogen-related molecular patterns, including phagocytosis and sterilization of phagocytes, antigen presentation of antigen presenting cells and the like. Therefore, substances targeting TLRs are thought to have immunomodulatory effects and may have potential therapeutic value. Several studies have now demonstrated the possibility of immunotherapeutic treatment of bacterial infections with agonists of TLRs. The agonists Imiquimod (Imiquimod) such as TLR7 and Resiquimod (Resiquimod) which is a TLR7/8 agonist have been used clinically for the treatment of viral infections. Recent studies have shown that TLRs ligands may also play a role in the treatment of bacterial infections. Such as synthetic TLR4 ligand AGPs, has protective effect on the infection of Listeria monocytogenes and Yersinia pestis; after the parasite-derived protein ES-62 is combined with TLR4, immune dysregulation in sepsis is improved by blocking a MyD 88/NF-kB signal channel, and the treatment effect is obvious; the agonist MALP-2 of TLR2/6 can enhance phagocytosis of macrophages, remarkably reduce mortality of mice co-infected with influenza virus-streptococcus pneumoniae and bacterial load of lungs, and has protective effect on pseudomonas aeruginosa infection.

However, no report has been made so far about the application of the double-ligand protein/polypeptide capable of binding to TLR2 and TLR4 to the treatment of bacterial infection diseases.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide an application of Toll-like receptor ligand protein in resisting bacterial infection, which is used for solving the problems of drug resistance, adverse side effects and the like existing in the existing therapeutic strategies for resisting bacterial infectious diseases.

In order to achieve the above objects and other related objects, the present invention provides a TLR2/4 dual ligand protein or polypeptide, wherein the protein or polypeptide is streptococcus pneumoniae endopeptidase O, the gene sequence of the protein or polypeptide is a polypeptide fragment represented by a nucleic acid sequence SEQ ID No.1, and the protein sequence of the protein or polypeptide is a polypeptide fragment represented by an amino acid sequence SEQ ID No. 2.

In a second aspect, the invention provides an isolated or purified protein or polypeptide as described above.

In a third aspect, the invention provides an isolated polynucleotide encoding a protein or polypeptide as described above.

Optionally, the sequence of the polynucleotide comprises at least one of:

(1) a fragment as set forth in SEQ ID No.1 or an RNA equivalent thereof;

(2) a sequence complementary to any of the sequences in (1);

(3) a sequence encoding the same protein or polypeptide as the sequence in (1) or (2);

(4) a fragment as set forth in SEQ ID No.2 or an RNA equivalent thereof;

(5) a sequence complementary to any of the sequences in (4);

(6) a sequence encoding the same protein or polypeptide as the sequence in (4) or (5);

in a fourth aspect, the invention provides a construct comprising the isolated polynucleotide described above.

In a fifth aspect, the invention provides an expression system comprising the above construct or a polynucleotide having an exogenous sequence integrated into the genome.

In a sixth aspect, the present invention provides a method for producing the above protein or polypeptide, comprising: culturing the expression system as described above under conditions suitable for expression of the protein or polypeptide.

In a seventh aspect, the invention provides an immunogenic and/or antigenic composition comprising a protein or polypeptide as described above.

The eighth aspect of the invention provides the application of the protein or polypeptide, the isolated polynucleotide, the construct and the expression system in preparing the medicines for preventing and/or treating the bacterial infection.

Optionally, the antibacterial infectious disease is at least one of bacterial pneumonia, bacterial meningitis, bacterial otitis media, bacteremia and sepsis.

Optionally, the medicament is at least one of respiratory nasal drops, intravenous injection and oral preparation.

As described above, the application of the Toll-like receptor ligand protein in resisting bacterial infection has the following beneficial effects: the protein (or polypeptide) is prepared by cloning and expressing streptococcus pneumoniae virulence genes, and the streptococcus pneumoniae endopeptidase O (PepO) is used for treating macrophages, so that the PepO can remarkably enhance the phagocytosis and killing effects of the macrophages on pathogenic bacteria by combining TLR2 and TLR4 on the surfaces of the macrophages, and can up-regulate the secretion of relevant cytokines and chemokines, thereby inducing strong innate immune response.

Drawings

FIG. 1 shows the results of PCR identification of recombinant plasmid pET28a (+) -PepO and nucleotide sequence identification (1893bp) of the protein PepO in example 1 of the present invention.

FIG. 2 is a graph showing the results of detection of purified expression (molecular weight: 72kDa) of PepO in example 2 of the present invention.

FIG. 3 is a graph showing the results of the measurement of the enhancement of phagocytosis of macrophages by the PepO protein in example 3 of the present invention.

FIG. 4 is a graph showing the results of measurement of the enhancement of the bactericidal effect of the PepO protein on macrophages in example 4 of the present invention.

FIG. 5 is a graph showing the results of detection of binding of recombinant PepO protein to TLR2 and TLR4 on the surface of mouse macrophage cells by co-immunoprecipitation (A) and detection of phagocytic function of each gene-deficient macrophage cell treated with PepO (B-D) in example 5 of the present invention.

FIG. 6 is a graph showing the results of the bacterial load after respiratory infection in mice in example 6 of the present invention.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The invention discloses an application of streptococcus pneumoniae endopeptidase O (PepO) in bacterial infection resistance, wherein the gene sequence is a nucleic acid sequence SEQ ID NO.1, and the protein sequence is an amino acid sequence SEQ ID NO. 2.

Since macrophages are one of the most important components of the innate immune response of the respiratory tract, they are the most abundant immune cells in lung homeostasis. Macrophages can enhance the body's ability to fight infection by two factors. On the one hand, the indirect effect is that the secretion of cytokines and chemokines promotes the recruitment of immune cells at the infected part, thereby enhancing the elimination of bacteria. On the other hand, the direct action is to directly remove pathogens through phagocytic killing function, thereby showing the anti-infection capability.

The invention discovers that Streptococcus pneumoniae endopeptidase O (PepO) can be identified by pattern recognition receptors TLR2 and TLR4 on the surface of macrophages, is combined with TLR2 and TLR4 on the surface of the macrophages and is combined with TLR2 and TLR4 on the surface of the macrophages, the phagocytosis of the macrophages is enhanced by up-regulating a surface receptor CR3 through miR155, autophagy is enhanced by regulating an Akt/mTOR signal channel, so that the phagocytosis and sterilization effects are enhanced, the secretion of cytokines and chemokines is increased, and strong innate immune response is induced, so that the elimination of pathogenic bacteria by a host is enhanced.

In the following examples, the preparation of a fusion protein vaccine for the prevention of streptococcus pneumoniae infection comprises the following steps:

(1) amplifying a PepO gene fragment from the genome of the streptococcus pneumoniae D39 strain by using a polymerase chain reaction method;

(2) constructing a pET28a (+) recombinant plasmid containing a PepO coding sequence;

(3) the recombinant plasmid is transformed into BL21(DE3) engineering bacteria, and IPTG induces high-efficiency expression of PepO recombinant protein;

(4) purifying the obtained recombinant protein by affinity chromatography;

(5) removing residual endotoxin in the purified recombinant protein.

Example 1

Construction of recombinant expression vector pET28a (+) -PepO.

Material (one):

prokaryotic expression plasmid pET28a (+) was purchased from Novagen, Prime Star Hi-Fi enzyme, dNTPs, Buffer, MgCl used for PCR₂From Bao bioengineering (Dalian) Ltd, PTC-200PCR instrument is Perkin Elmer product.

(II) design and synthesis of primers:

the primers were designed using premier5.0 with reference to the entire sequence of genomic DNA of Streptococcus pneumoniae D39 (GeneBank accession No. CP000410.2) as a template, and synthesized by Biotechnology engineering (Shanghai) GmbH.

PepO: an upstream primer: 5'-GCCATGGCACGTTATCAAGATGATTTTTAT-3', containing an NcoI site;

a downstream primer: 5'-CCCTCGAGCCAAATAATCACGCGCTCCTCT-3', containing an XhoI site.

(III) PCR amplification of the target gene:

and (3) amplification of the PepO gene, wherein the nucleotide sequence of the amplified PepO gene is shown as SEQ ID NO. 1.

An amplification system:

wherein "P1 (5pM) 2. mu.l" means: taking a PepO upstream primer with the concentration of 5pM, and adding 2 mul;

"P2 (5pM) 2. mu.l" means: the PepO downstream primer with a concentration of 5pM was added in an amount of 2. mu.l.

Conditions are as follows: 2min at 98 ℃; 45s at 55 ℃, 90s at 72 ℃ and 33 cycles; 10min at 72 ℃ for 1 time. The corresponding target gene is obtained by amplification under the above conditions.

(IV) construction of prokaryotic expression vector

The recovery of PCR products was carried out according to the kit instructions supplied by Roche, and the plasmid pET28a (+) was carried out according to the instruction of the Omega miniplasmid DNA extraction kit. The vector DNA and the PepO gene were digested simultaneously with NcoI and BamH1, and then recovered and purified by Roche kit. The enzyme digestion reaction system is as follows:

after the reaction system is placed in a water bath kettle at 37 ℃ for reaction for 3h, products are identified by agarose gel electrophoresis and are cut by a Roche kit to recover the products. The digested PepO gene fragment and the pET28a (+) plasmid were ligated by T4 ligase in the following reaction system:

the reaction system was placed in a 22 ℃ water bath and connected for 1 h.

(V) transformation and identification of the ligation products:

slowly melting 200 μ L DH5 α stored at-80 deg.C in ice bath, adding 10 μ L ligation reaction product, gently mixing, and performing ice water bath for 30 min; heat shock is carried out for 30s at 42 ℃, and then ice water bath is carried out for 2 min; adding 800 mu lLB culture medium into the bacterial liquid, culturing at 37 ℃ and 180rpm for 1 h; centrifuging the bacterial liquid at 5000rpm for 5min, discarding 800 μ L, mixing the rest 200 μ L bacterial liquid, coating on LK plate, and incubating overnight at 37 deg.C.

20 single colonies are picked, PCR identification is carried out by using PepO upstream and downstream primers (the reaction system and conditions are as described above), 5 PCR positive colonies are selected for enrichment culture, and then the colonies are sent to the department of Onychidae for sequencing identification. Transformants with the correct sequencing were kept for use.

Example 2

Expression, identification and purification of prokaryotic expression plasmid pET28a (+) -PepO in escherichia coli

The recombinant plasmid pET28a (+) -PepO is transformed into Escherichia coli competent BL21(DE 3). The process and conditions were as described in example 1. And (4) sequencing and identifying the positive transformants and storing for later use.

Secondly, IPTG induces the mass expression of the PepO recombinant protein.

The preserved pET28a (+) -PepO-BL21(DE3) Escherichia coli was added to 30mL of LB medium, cultured at 37 ℃ and 180rpm for 8 hours, and then all the bacterial solutions were transferred to 500mL of LB medium and cultured for 4 hours at 37 ℃ and 180 rpm. IPTG was then added to the final concentration of 40mM in the broth and the cells were incubated at 22 ℃ and 180rpm overnight.

And (III) purifying the recombinant protein.

Centrifuging at the temperature of 4 ℃ and 12000g for 10min, and then carrying out ultrasonic bacteria breaking after resuspension by using 30 mLbindingbuffer; after 12000g of the bacterial disruption solution is processed at 4 ℃ for 10min, supernatant is separated, the supernatant is filtered by a 0.45 mu m filter membrane, and filtrate is collected for later use.

Affinity chromatography purification: 2ml of 50% Ni were aspirated²⁺-the NTA resin suspension is passed through the resin twice equilibrated in a column with 20mL of biningbuffer; ni after equilibrium²⁺-mixing NTA resin suspension with the above filtrate, and incubating at 4 deg.C for 1h with slow shaking; transferring the suspension into a chromatographic column, allowing the liquid to naturally flow out, and balancing a column bed; gradient elution is carried out on different imidazole concentrations, and the eluates are respectively collected.

And (IV) identifying the eluates of imidazole with different concentrations by SDS-PAGE and Coomassie brilliant blue staining, selecting the eluent with the highest PepO purity and abundance, and performing pressurized ultrafiltration by using an ultrafiltration membrane with 20kD and a PBS buffer solution with the pH value of 8.0 to remove the imidazole in the protein. The protein after ultrafiltration is detected by SDS-PAGE and Coomassie brilliant blue, and the concentration is over 95 percent.

And (V) removing residual endotoxin in the recombinant protein. Residual endotoxin in the protein was removed using an endotoxin removal kit provided by the Kinsley company, and the operation method and the steps were performed according to the manufacturer's instructions.

(V) quantification of recombinant protein (BCA method)

The concentration of the recombinant protein was determined using a Biyuntian kit, and the methods of operation and steps were performed according to the manufacturer's instructions.

Example 3

Promotion of phagocytosis of macrophages by PepO

And (I) culturing streptococcus pneumoniae. Streptococcus pneumoniae D39 strain (NCTC 7466) was derived from the American type culture Collection (NCTC). Inoculating cryopreserved bacteria on Columbia blood plate, and culturing at 37 deg.C under 5% CO₂Incubated under conditions overnight. The monoclonal colonies were inoculated into 10ml of C + Y medium at 37 ℃ with 5% CO₂Culturing until the bacteria is in middle logarithmic growth phase (OD)₆₀₀)＝0.5。

(II) extracting and processing macrophages. C57/BL6 mice of 8 weeks old are selected, injected with 1mL of sterile paraffin oil in the abdominal cavity, the mice are killed after being broken neck after 96 hours, and the abdominal cavity of the mice is irrigated twice with precooled PBS buffer solution containing 0.1% EDTA-Na, wherein each time is 8 mL. The lavage fluid was centrifuged at 800g for 5min to remove paraffin oil and supernatant, red blood cells were removed with red blood cell lysate, and then resuspended in DMEM high-sugar medium and counted, followed by plating into 9cm dishes (10 per dish)⁷Individual cells). And (5) after the plates are paved for 45min, changing the liquid to remove the cells which are not attached to the wall, wherein the attached cells are the mouse macrophages. After overnight incubation, macrophages were treated with PepO (10. mu.g/mL) for 6 hours before bacterial infection.

(III) Streptococcus pneumoniae D39 infects macrophages.

Culturing Streptococcus pneumoniae D39 to logarithmic metaphase, centrifuging (8000rpm, 5min), collecting bacteria, washing with sterile PBS buffer solution twice, and diluting with DMEM high-sugar medium to desired concentration (5 × 10)⁷CFU/mL). After the treated macrophage is subjected to liquid change, adding the bacterial liquid into a 24-pore plate, incubating for 30min, washing for 3 times by using sterile PBS buffer, and addingGentamicin (200. mu.g/mL) was incubated for 10min and then washed 5 more times to remove extracellular bacteria. Sterile water was then added for 10min incubation and scraped with a pipette tip to disrupt the cells.

And (IV) counting the planks.

The cell lysate was diluted 10-, 100-and 1000-fold with sterile PBS buffer, mixed well and plated 10 μ L to columbia blood plates (3 spots per dilution were averaged). 37 ℃ and 5% CO₂After overnight incubation, the number of colonies formed was counted and statistically analyzed.

(V) the different types of bacteria phagocytosed by the mouse macrophages treated with the recombinant PepO protein at different concentrations are shown in FIG. 3, wherein A is Streptococcus pneumoniae, B is Staphylococcus aureus, and C is Pseudomonas aeruginosa. The results show that PepO can significantly enhance phagocytosis of macrophages, and enhance phagocytosis of gram-positive bacteria, namely streptococcus pneumoniae and staphylococcus aureus, and gram-negative bacteria, namely pseudomonas aeruginosa, which indicates that the effect is independent of the type of bacteria.

Example 4

Enhancement of macrophage bactericidal effect by PepO

(I) culturing bacteria. The culture method and conditions of staphylococcus aureus and pseudomonas aeruginosa were the same as those of streptococcus pneumoniae in example 4.

(II) extracting, culturing and processing the macrophages. This part is the same as example 3.

(III) Streptococcus pneumoniae D39 infects macrophages.

Culturing Streptococcus pneumoniae D39 to logarithmic metaphase, centrifuging (8000rpm, 5min), collecting bacteria, washing with sterile PBS buffer solution twice, and diluting with DMEM high-sugar medium to desired concentration (5 × 10)⁷CFU/mL). The bacterial solution was added to a 24-well plate and incubated for 120min, washed 3 times with sterile PBS buffer, added with gentamicin (200. mu.g/mL), incubated for 10min and washed 5 times to remove extracellular bacteria. Sterile water was then added for 10min incubation and scraped with a pipette tip to disrupt the cells.

And (IV) counting the planks. This part is the same as example 3.

(V) the killing effect of the mouse macrophage treated by the recombinant PepO protein with different concentrations on different kinds of bacteria is shown in figure 4, wherein A is streptococcus pneumoniae, B is staphylococcus aureus and C is pseudomonas aeruginosa. The result shows that PepO can obviously enhance the bactericidal action of macrophages, and enhances the bactericidal action on gram-positive bacteria, namely streptococcus pneumoniae and staphylococcus aureus, and gram-negative bacteria, namely pseudomonas aeruginosa, which indicates that the action is independent of the types of bacteria.

Example 5

PepO promotes macrophage function and enhances host anti-infection capacity through TLR2 and TLR4

(I) detection of PepO binding to mouse TLR2 and TLR4 by co-immunoprecipitation

Extraction and culture of mouse macrophages were performed as described above, and after changing the medium, recombinant PepO protein (10. mu.g/mL) was added to the culture dish and treated for 1 hour. After the treatment, the medium was discarded and the cells were washed twice with pre-cooled PBS, then 1mL of non-denatured protein lysate was added, the cells were collected with a pre-cooled cell scraper, the cell suspension was transferred to a 1.5mL EP tube, after shaking slowly at 4 ℃ for 15 minutes, centrifuged at 14000g at 4 ℃ for 30 minutes and the supernatant was immediately collected. The Protein A agarose beads were washed twice with PBS and diluted to 50%, and after adding 100. mu.L of agarose beads per 1mL of cell lysate supernatant, they were gently shaken at 4 ℃ for 10 minutes to remove non-specific binding. After centrifugation at 14000g for 15 minutes at 4 ℃ the supernatant was collected and the agarose beads were removed. The rabbit anti-FLAG tag antibody was added to the total Protein and shaken slowly overnight at 4 ℃ before adding 100 μ L Protein a agarose beads and shaking slowly at 4 ℃ overnight to capture the antigen-antibody complex. The antigen-antibody complex was then collected by centrifugation at 14000g for 5s at 4 ℃.

The antigen-antibody complex was washed three times with PBS, and then 3X Loading Buffer was added, and after boiling for 5 minutes, the protein was separated by 10% SDS-PAGE. And electrically transferring the separated protein onto a PVDF membrane, sealing the PVDF membrane by using 5% skimmed milk powder, incubating corresponding primary antibody overnight, incubating corresponding secondary antibody, developing by using an ECL method, and detecting the protein.

The results of detection of binding of Pep0 to TLR2 and TLR4 on the macrophage surface are shown in a in fig. 5. This result demonstrates that recombinant Pep0 protein can interact directly with TLR2 and TLR 4.

The promotion of the phagocytic function of the macrophage by the Pep0 depends on the TLR2 and TLR4

The macrophage extraction, culture and treatment and infection experiment of the TLR2 gene deficient mouse, the TLR4 gene deficient mouse and the TLR2/4 double gene deficient mouse are the same as the example 3. The experiments for Pep0 treatment and Streptococcus pneumoniae infection of TLR2/4 double gene deficient mice were the same as in example 3. The TLR2 gene-deficient mouse and TLR4 gene-deficient mouse are purchased from JacksonLab in the United states, and the TLR2/4 double-gene-deficient mouse is derived from the hybridization of TLR2 gene-deficient mouse and TLR4 gene-deficient mouse; culturing and breeding the 3 gene-deficient mice in the experimental animal center of Chongqing university of medicine; before the experiment, the reliability of the experiment is ensured by identifying through a genotype identification method provided by manufacturers.

The results of the detection of the phagocytic function of each gene-deficient macrophage after the PepO treatment are shown in fig. 5B to D. Wherein FIGS. 5-B and 5-C show the amount of Streptococcus pneumoniae phagocytosed by TLR 2-and TLR 4-deficient macrophages after stimulation by PepO at different concentrations, respectively, it can be seen that the deficient cells require higher doses than wild-type cells, but PepO can still enhance phagocytosis by macrophages; FIG. 5-D shows the lung bacterial load of the mice with double gene defects of TLR2/4 treated by PepO after being infected with Streptococcus pneumoniae, which is shown in no response to the stimulation of PepO, and thus PepO can not reduce the lung bacterial load of the mice with double gene defects of TLR 2/4. The results show that the enhancement of macrophage function and host anti-infection defense effect by PepO depends on TLR2 and TLR 4.

Example 6

Protection effect of PepO on respiratory tract infection of mice

Grouping of mice.

Female C57/BL6 mice were randomly divided into 3 groups of similar body weight, a blank control group (treated with PBS), an irrelevant control group (treated with another streptococcus pneumoniae recombinant protein LytR prepared in the same manner), and an experimental group (treated with PepO recombinant protein), each of which was 6 mice. For s.aureus infected mice, no independent control group was set, and the experimental group contained 12 mice for testing the bacterial load at 24 hours and 48 hours after infection.

And (II) treating the mice.

The recombinant protein concentration was adjusted with sterile PBS to equalize the molarity between LytR and PepO (3.5. mu.M). Each mouse was given 40. mu.L (20. mu.L per nostril) of sterile PBS, LytR or PepO nasally.

(III) infection of mice.

After 6 hours of treatment, the nasal drops were infected with Streptococcus pneumoniae D39 strain (1X 10)⁷Only).

And (IV) after 24 hours of infection, anesthetizing the mice, taking blood and lung homogenate, diluting, plating and counting bacterial load.

And (V) treating the mice by adopting the same method and infecting pseudomonas aeruginosa, anaesthetizing the mice after 24 hours, taking blood and lung homogenate, diluting, and plating and counting the bacterial load.

And (VI) treating the mice by adopting the same method, infecting staphylococcus aureus, anesthetizing 6 mice respectively after 24 hours and 48 hours, taking nasal cavity lavage fluid, blood and lung homogenate, diluting, and plating and counting the bacterial load.

(VII) the bacterial load after respiratory infection of mice is shown in figure 6, wherein A is Streptococcus pneumoniae and B is Pseudomonas aeruginosa. The results show that the lung capacity of mice in the experimental group treated with PepO nasal drops was significantly reduced compared to the blank control group, whereas the lung capacity of mice was not reduced by the streptococcus pneumoniae recombinant protein LytR in the unrelated control group. In fig. 6, C and D are the bacterial load in the mouse lung and nasal lavage fluid 24 hours after staphylococcus aureus infection, respectively, and E and F are the bacterial load in the mouse lung and nasal lavage fluid 48 hours after staphylococcus aureus infection, respectively. The results show that PepO treatment not only reduced lung loading in mice, but also reduced the bacteria colonized in the nasal cavity.

By combining the results, the PepO can obviously reduce the bacterial load of gram-positive bacteria streptococcus pneumoniae, staphylococcus aureus and gram-negative bacteria pseudomonas aeruginosa in respiratory tracts, which indicates that the effect is irrelevant to the type of infected bacteria.

Through the above experimental results, it can be found that: the invention successfully prepares a novel immunomodulator PepO, explains the action mode of the immunomodulator PepO and evaluates the respiratory tract infection protection effect of the immunomodulator PepO. The result shows that the PepO can interact with TLR2 and TLR4 on the surface of macrophage, thereby obviously enhancing the phagocytosis and killing effect of the macrophage on different pathogenic bacteria, obviously increasing the bacterial clearance of the respiratory tract of a mouse and enhancing the anti-infection capability of the mouse. As a recombinant protein, PepO has the advantages of single component, simple and convenient preparation, easy standardization, low toxicity and the like, and is an immunomodulator with application potential.

In conclusion, the streptococcus pneumoniae endopeptidase (PepO) has an immunomodulator-like effect, can be used for enhancing the immune response and the anti-infection capacity of an organism and can effectively resist respiratory tract infection of various pathogenic bacteria.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

SEQUENCE LISTING

<110> Chongqing university of medical science

<120> application of Toll-like receptor ligand protein in bacterial infection resistance

<130>PCQYK197799

<160>3

<170>PatentIn version 3.5

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<211>1893

<212>DNA

<213>Artificial

<220>

<223> PepO nucleotide sequence

<400>1

atgacacgtt atcaagatga tttttatgat gctatcaatg gagaatggca acagacagct 60

gaaatcccag cagataagtc tcaaacagga ggttttgttg atttagacca ggaaattgaa 120

gacctgatgt tggcgacaac agacaagtgg ttagcaggtg aagaagtgcc tgaggatgct 180

atcttggaaa actttgtcaa ataccaccgc ctagttcgtg attttgacaa gagagaagct 240

gacggtatca cacctgtctt accactcctt aaagaattcc aagaattgga aacttttgcg 300

gattttacag ctaaactagc agagtttgag cttgcaggaa aaccaaactt ccttcctttt 360

ggtgtatcgc cagactttat ggatgctaga atcaatgttc tatgggctag cgctccaagc 420

acaatcttgc cagatacgac ctactatgca gaagaacatc ctcagcgcga agagctcttg 480

actctttgga aagaaagcag cgcaaatctc ctcaaggctt atgatttctc tgatgaagaa 540

attgaagact tgctagaaaa aagacttgaa ttggaccgcc gagttgcggc agtggtgctc 600

tctaatgaag aaagttcaga atatgctaaa ctctatcatc catattctta cgaagatttc 660

aagaaattcg cgcctgccct acctttggat gacttcttca aagcagttat tgggcaatta 720

ccagacaagg ttattgtaga cgaggaacgt ttctggcaag cagcagagca attctacagt 780

gaggaatcct ggtctctcct taaagcaacc ttgattttga gtgttgtcaa tctttcaacc 840

agctatttaa cagaggatat ccgtgttttg tctggtgcct acagccgtgc cctttctgga 900

gttccagagg caaaagataa ggtcaaagca gcttatcatc tagcacaaga acctttcaag 960

caagccctgg ggctttggta cgcccgtgag aagttctctc cagaagccaa ggcggatgtg 1020

gagaaaaaag tggcaaccat gattgatgtc tataaggagc gtctgcttaa gaatgactgg 1080

ctcactccag aaacctgtaa acaggctatc gtgaagctca atgtgatcaa accttatatt 1140

ggctatccag aagaattgcc tgcacgttac aaggataagg tagtgaatga aactgccagt 1200

ctttttgaga atgctctagc ctttgcgcgt gtggaaatca agcacagttg gagtaagtgg 1260

aaccagcctg tagactataa ggaatggggc atgcctgctc atatggtcaa tgcctactac 1320

aatcctcaga agaacctgat tgtctttcca gcggccattt tacaggcgcc tttctatgac 1380

ttgcatcagt catcttctgc taactacggt ggtattgggg cagtgattgc ccatgaaatt 1440

tcccacgcct ttgatactaa cggggcttcc tttgacgaaa atggtagcct caaggattgg 1500

tggacagaga gcgactatgc tgccttcaag gagaaaacac aaaaagtcat tgaccaattt 1560

gatggacagg attcttatgg agcaaccatt aacggtaaat tgactgtatc agaaaacgtg 1620

gctgacttgg gaggaatcgc agcagcgctt gaagcagcta agagagaagc agacttctca 1680

gcagaagagt tcttctacaa cttcggtcgc atctggcgca tgaaaggtcg tccagaattt 1740

atgaaacttt tggctagcgt cgatgtgcac gcaccagcca aactccgtgt caatgtgcaa 1800

gtaccaaact tcgacgattt ctttacaacc tatgatgtca aagaaggaga cggaatgtgg 1860

cgttcaccag aggagcgcgt gattatttgg taa 1893

<210>2

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<223> PepO amino acid sequence

<400>2

Met Thr Arg Tyr Gln Asp Asp Phe Tyr Asp Ala Ile Asn Gly Glu Trp

1 5 10 15

Gln Gln Thr Ala Glu Ile Pro Ala Asp Lys Ser Gln Thr Gly Gly Phe

20 25 30

Val Asp Leu Asp Gln Glu Ile Glu Asp Leu Met Leu Ala Thr Thr Asp

35 40 45

Lys Trp Leu Ala Gly Glu Glu Val Pro Glu Asp Ala Ile Leu Glu Asn

50 55 60

Phe Val Lys Tyr His Arg Leu Val Arg Asp Phe Asp Lys Arg Glu Ala

65 70 75 80

Asp Gly Ile Thr Pro Val Leu Pro Leu Leu Lys Glu Phe Gln Glu Leu

85 90 95

Glu Thr Phe Ala Asp Phe Thr Ala Lys Leu Ala Glu Phe Glu Leu Ala

100 105 110

Gly Lys Pro Asn Phe Leu Pro Phe Gly Val Ser Pro Asp Phe Met Asp

115 120 125

Ala Arg Ile Asn Val Leu Trp Ala Ser Ala Pro Ser Thr Ile Leu Pro

130 135 140

Asp Thr Thr Tyr Tyr Ala Glu Glu His Pro Gln ArgGlu Glu Leu Leu

145 150 155 160

Thr Leu Trp Lys Glu Ser Ser Ala Asn Leu Leu Lys Ala Tyr Asp Phe

165 170 175

Ser Asp Glu Glu Ile Glu Asp Leu Leu Glu Lys Arg Leu Glu Leu Asp

180 185 190

Arg Arg Val Ala Ala Val Val Leu Ser Asn Glu Glu Ser Ser Glu Tyr

195 200 205

Ala Lys Leu Tyr His Pro Tyr Ser Tyr Glu Asp Phe Lys Lys Phe Ala

210 215 220

Pro Ala Leu Pro Leu Asp Asp Phe Phe Lys Ala Val Ile Gly Gln Leu

225 230 235 240

Pro Asp Lys Val Ile Val Asp Glu Glu Arg Phe Trp Gln Ala Ala Glu

245 250 255

Gln Phe Tyr Ser Glu Glu Ser Trp Ser Leu Leu Lys Ala Thr Leu Ile

260 265 270

Leu Ser Val Val Asn Leu Ser Thr Ser Tyr Leu Thr Glu Asp Ile Arg

275 280 285

Val Leu Ser Gly Ala Tyr Ser Arg Ala Leu Ser Gly Val Pro Glu Ala

290 295 300

Lys Asp Lys Val Lys Ala Ala Tyr His Leu Ala Gln Glu ProPhe Lys

305 310 315 320

Gln Ala Leu Gly Leu Trp Tyr Ala Arg Glu Lys Phe Ser Pro Glu Ala

325 330 335

Lys Ala Asp Val Glu Lys Lys Val Ala Thr Met Ile Asp Val Tyr Lys

340 345 350

Glu Arg Leu Leu Lys Asn Asp Trp Leu Thr Pro Glu Thr Cys Lys Gln

355 360 365

Ala Ile Val Lys Leu Asn Val Ile Lys Pro Tyr Ile Gly Tyr Pro Glu

370 375 380

Glu Leu Pro Ala Arg Tyr Lys Asp Lys Val Val Asn Glu Thr Ala Ser

385 390 395 400

Leu Phe Glu Asn Ala Leu Ala Phe Ala Arg Val Glu Ile Lys His Ser

405 410 415

Trp Ser Lys Trp Asn Gln Pro Val Asp Tyr Lys Glu Trp Gly Met Pro

420 425 430

Ala His Met Val Asn Ala Tyr Tyr Asn Pro Gln Lys Asn Leu Ile Val

435 440 445

Phe Pro Ala Ala Ile Leu Gln Ala Pro Phe Tyr Asp Leu His Gln Ser

450 455 460

Ser Ser Ala Asn Tyr Gly Gly Ile Gly Ala Val Ile Ala His Glu Ile

465 470 475 480

Ser His Ala Phe Asp Thr Asn Gly Ala Ser Phe Asp Glu Asn Gly Ser

485 490 495

Leu Lys Asp Trp Trp Thr Glu Ser Asp Tyr Ala Ala Phe Lys Glu Lys

500 505 510

Thr Gln Lys Val Ile Asp Gln Phe Asp Gly Gln Asp Ser Tyr Gly Ala

515 520 525

Thr Ile Asn Gly Lys Leu Thr Val Ser Glu Asn Val Ala Asp Leu Gly

530 535 540

Gly Ile Ala Ala Ala Leu Glu Ala Ala Lys Arg Glu Ala Asp Phe Ser

545 550 555 560

Ala Glu Glu Phe Phe Tyr Asn Phe Gly Arg Ile Trp Arg Met Lys Gly

565 570 575

Arg Pro Glu Phe Met Lys Leu Leu Ala Ser Val Asp Val His Ala Pro

580 585 590

Ala Lys Leu Arg Val Asn Val Gln Val Pro Asn Phe Asp Asp Phe Phe

595 600 605

Thr Thr Tyr Asp Val Lys Glu Gly Asp Gly Met Trp Arg Ser Pro Glu

610 615 620

Glu Arg Val Ile Ile Trp

625630

<210>3

<211>1017

<212>PRT

<213>Artificial

<220>

<223> recombinant protein LytR amino acid sequence

<400>1

Ala Thr Gly Gly Thr Thr Ala Ala Ala Ala Ala Ala Ala Thr Thr Ala

1 5 10 15

Thr Thr Gly Gly Ala Ala Thr Gly Gly Thr Gly Cys Thr Ala Gly Cys

20 25 30

Thr Thr Thr Ala Cys Thr Thr Thr Cys Thr Gly Thr Ala Ala Cys Thr

35 40 45

Gly Thr Ala Gly Thr Ala Gly Gly Ala Gly Thr Ala Gly Gly Thr Gly

50 55 60

Thr Thr Thr Thr Thr Gly Cys Thr Thr Ala Thr Ala Cys Thr Ala Thr

65 70 75 80

Thr Thr Ala Thr Cys Ala Ala Cys Ala Ala Gly Gly Gly Ala Cys Ala

85 90 95

Gly Ala Ala Ala Cys Cys Thr Thr Ala Gly Cys Thr Ala Ala Ala Ala

100 105 110

Cys Cys Thr Ala Thr Ala Ala Ala Ala Ala Ala Ala Thr Cys Gly Gly

115120 125

Thr Gly Ala Ala Gly Ala Ala Ala Cys Cys Ala Ala Gly Gly Thr Thr

130 135 140

Ala Thr Thr Gly Ala Ala Gly Cys Gly Ala Cys Thr Gly Ala Ala Cys

145 150 155 160

Cys Thr Cys Thr Ala Ala Cys Cys Ala Thr Thr Cys Thr Gly Thr Thr

165 170 175

Ala Ala Thr Gly Gly Gly Ala Gly Thr Gly Gly Ala Cys Ala Cys Cys

180 185 190

Gly Gly Ala Ala Ala Thr Gly Thr Thr Gly Ala Ala Cys Gly Ala Ala

195 200 205

Cys Thr Gly Ala Ala Ala Cys Thr Thr Gly Gly Gly Thr Cys Gly Gly

210 215 220

Thr Ala Gly Ala Ala Gly Thr Gly Ala Thr Ala Gly Cys Ala Thr Gly

225 230 235 240

Ala Thr Cys Thr Thr Gly Ala Thr Gly Ala Cys Ala Gly Thr Gly Ala

245 250 255

Ala Thr Cys Cys Thr Ala Ala Ala Ala Cys Gly Ala Ala Ala Ala Ala

260 265 270

Ala Ala Cys Ala Ala Cys Ala Ala Thr Gly Ala Thr Gly Ala Gly Thr

275 280 285

Thr Thr Ala Gly Ala Gly Cys Gly Gly Gly Ala Thr Ala Thr Thr Cys

290 295 300

Thr Gly Ala Cys Gly Cys Gly Cys Ala Thr Thr Gly Ala Ala Thr Cys

305 310 315 320

Ala Gly Gly Gly Ala Ala Thr Gly Gly Thr Cys Ala Gly Gly Cys Thr

325 330 335

Cys Ala Thr Gly Ala Ala Gly Cys Gly Ala Ala Ala Cys Thr Gly Ala

340 345 350

Ala Cys Thr Cys Ala Gly Cys Ala Thr Ala Thr Gly Cys Ala Gly Ala

355 360 365

Thr Gly Gly Thr Gly Gly Ala Gly Cys Ala Gly Ala Gly Cys Thr Thr

370 375 380

Gly Cys Thr Ala Thr Ala Gly Ala Ala Ala Cys Cys Ala Thr Thr Cys

385 390 395 400

Ala Ala Ala Ala Ala Ala Thr Gly Ala Thr Gly Ala Ala Thr Ala Thr

405 410 415

Cys Cys Ala Thr Ala Thr Thr Gly Ala Thr Cys Gly Cys Thr Ala Thr

420 425 430

Gly Thr Gly Ala Thr Gly Gly Thr Cys Ala Ala Thr Ala Thr Gly Ala

435 440445

Gly Ala Gly Gly Gly Thr Thr Gly Cys Ala Ala Ala Ala Ala Thr Thr

450 455 460

Ala Gly Thr Gly Gly Ala Thr Gly Cys Ala Gly Thr Ala Gly Gly Ala

465 470 475 480

Gly Gly Thr Ala Thr Thr Ala Cys Ala Gly Thr Cys Ala Ala Thr Ala

485 490 495

Ala Thr Ala Thr Cys Cys Thr Ala Gly Gly Thr Thr Thr Cys Cys Cys

500 505 510

Ala Ala Thr Thr Thr Cys Thr Ala Thr Cys Ala Gly Thr Gly Ala Cys

515 520 525

Cys Ala Ala Gly Ala Ala Gly Ala Ala Thr Thr Thr Ala Ala Thr Ala

530 535 540

Cys Cys Ala Thr Thr Thr Cys Thr Ala Thr Cys Gly Gly Thr Gly Thr

545 550 555 560

Thr Gly Gly Gly Gly Ala Gly Cys Ala Ala Cys Ala Thr Ala Thr Thr

565 570 575

Gly Gly Gly Gly Gly Ala Gly Ala Ala Gly Ala Ala Gly Cys Cys Cys

580 585 590

Thr Ala Gly Thr Cys Thr Ala Thr Gly Cys Ala Cys Gly Ala Ala Thr

595 600605

Gly Cys Gly Thr Thr Ala Cys Cys Ala Ala Gly Ala Thr Cys Cys Thr

610 615 620

Gly Ala Gly Gly Gly Gly Gly Ala Thr Thr Ala Thr Gly Gly Thr Cys

625 630 635 640

Gly Thr Cys Ala Ala Ala Ala Ala Cys Gly Thr Cys Ala Ala Cys Gly

645 650 655

Thr Gly Ala Ala Gly Thr Thr Ala Thr Thr Cys Ala Ala Ala Ala Ala

660 665 670

Gly Thr Cys Ala Thr Gly Gly Ala Ala Ala Ala Ala Gly Cys Thr Cys

675 680 685

Thr Cys Ala Gly Thr Thr Thr Ala Ala Ala Thr Ala Gly Cys Ala Thr

690 695 700

Thr Gly Gly Thr Cys Ala Thr Thr Ala Thr Cys Ala Ala Gly Ala Gly

705 710 715 720

Ala Thr Thr Cys Thr Ala Ala Ala Ala Gly Cys Thr Thr Thr Gly Ala

725 730 735

Gly Thr Gly Ala Cys Ala Ala Thr Ala Thr Gly Cys Ala Gly Ala Cys

740 745 750

Cys Ala Ala Thr Ala Thr Thr Gly Ala Thr Thr Thr Gly Thr Cys Thr

755 760 765

Gly Cys Ala Ala Ala Ala Ala Gly Thr Ala Thr Cys Cys Cys Thr Ala

770 775 780

Ala Cys Thr Thr Gly Cys Thr Ala Gly Gly Cys Thr Ala Thr Ala Ala

785 790 795 800

Ala Gly Ala Thr Thr Cys Ala Thr Thr Thr Ala Ala Ala Ala Cys Cys

805 810 815

Ala Thr Thr Gly Ala Ala Ala Cys Thr Cys Ala Gly Cys Ala Gly Thr

820 825 830

Thr Gly Cys Ala Gly Gly Gly Thr Gly Ala Ala Gly Gly Ala Gly Ala

835 840 845

Gly Ala Thr Ala Cys Thr Thr Cys Ala Ala Gly Gly Thr Gly Thr Thr

850 855 860

Thr Cys Thr Thr Ala Cys Cys Ala Gly Ala Thr Thr Gly Thr Thr Thr

865 870 875 880

Cys Gly Ala Gly Ala Gly Cys Ala Cys Ala Thr Ala Thr Gly Thr Thr

885 890 895

Gly Gly Ala Ala Ala Thr Gly Cys Ala Ala Ala Ala Thr Ala Thr Ala

900 905 910

Cys Thr Cys Cys Gly Ala Cys Gly Thr Thr Cys Thr Thr Thr Gly Gly

915 920 925

Gly Ala Cys Ala Ala Gly Ala Ala Gly Ala Ala Gly Thr Thr Ala Cys

930 935 940

Thr Cys Ala Gly Cys Thr Thr Gly Ala Ala Ala Cys Cys Ala Ala Thr

945 950 955 960

Gly Cys Gly Gly Thr Thr Thr Thr Ala Thr Thr Thr Gly Ala Ala Gly

965 970 975

Ala Thr Thr Thr Ala Thr Thr Thr Gly Gly Cys Ala Gly Ala Gly Cys

980 985 990

Ala Cys Cys Thr Gly Thr Thr Gly Gly Thr Gly Ala Thr Gly Ala Ala

995 1000 1005

Gly Ala Thr Ala Ala Thr Thr Ala Ala

1010 1015

Claims (10)

1. A TLR2/4 biligand protein or polypeptide, wherein: the protein or the polypeptide is streptococcus pneumoniae endopeptidase O, the gene sequence of the protein or the polypeptide is a polypeptide fragment shown by a nucleic acid sequence SEQ ID NO.1, and the protein sequence is a polypeptide fragment shown by an amino acid sequence SEQ ID NO. 2.

2. An isolated or purified protein or polypeptide of claim 1.

3. An isolated polynucleotide encoding the protein or polypeptide of claim 1 or 2.

4. The polynucleotide of claim 3, wherein: the sequence of the polypeptide comprises at least one of the following sequences:

(1) a fragment as set forth in SEQ ID No.1 or an RNA equivalent thereof;

(2) a sequence complementary to any of the sequences in (1);

(3) a sequence encoding the same protein or polypeptide as the sequence in (1) or (2);

(4) a fragment as set forth in SEQ ID No.2 or an RNA equivalent thereof;

(5) a sequence complementary to any of the sequences in (4);

(6) a sequence encoding the same protein or polypeptide as the sequence in (4) or (5).

5. A construct comprising the isolated polynucleotide of claim 3.

6. An expression system comprising the construct or genome of claim 5 having integrated therein an exogenous polynucleotide of claim 4.

7. A method for producing a protein or polypeptide according to claim 1 or 2, comprising: culturing the expression system of claim 6 under conditions suitable for expression of the protein or polypeptide.

8. An immunogenic and/or antigenic composition characterized by: the composition comprises the protein or polypeptide of claim 1 or 2.

9. Use of the protein or polypeptide according to claim 1 or 2, the isolated polynucleotide according to claim 3 or 4, the construct according to claim 5, or the expression system according to claim 6 for the preparation of a medicament for the prevention and/or treatment of an antibacterial infectious disease.

10. The use according to claim 10, wherein the antibacterial infectious disease is at least one of bacterial pneumonia, bacterial meningitis, bacterial otitis media, bacteremia, and sepsis;

and/or the medicine is at least one of respiratory tract nasal drops, intravenous injection and oral preparations.

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