CN108129555B

CN108129555B - Design of polypeptide specifically binding to immune protein of pseudomonas aeruginosa hexa-type secretion system and verification of antibacterial activity of polypeptide

Info

Publication number: CN108129555B
Application number: CN201710811434.3A
Authority: CN
Inventors: 崔胜�; 高小攀; 牟志霞; 秦博
Original assignee: Institute of Pathogen Biology of CAMS
Current assignee: Institute of Pathogen Biology of CAMS
Priority date: 2017-09-11
Filing date: 2017-09-11
Publication date: 2020-04-28
Anticipated expiration: 2037-09-11
Also published as: CN108129555A

Abstract

The invention discloses design of a polypeptide specifically combined with pseudomonas aeruginosa hexa-type secretion system immune protein and verification of antibacterial activity of the polypeptide. The invention firstly protects a polypeptide which is shown as a sequence 3 in a sequence table or a polypeptide which is shown as a sequence 4 in the sequence table. The gene encoding the polypeptide also belongs to the protection scope of the invention. The invention also protects the application of the polypeptide: binding to an immunity protein in a bacterial type vi secretion system; enriching immune proteins in a bacterial type VI secretion system; and (3) detecting immune protein in the bacterial VI type secretion system. The functional short peptide provided by the invention can be competitively combined with an effector protein to an immune protein, so that the TpLE-TpLEi interaction is damaged, the effector protein is promoted to crack the cell membrane of the bacterium to cause the bacterium to die, the novel antibacterial strategy which takes the T6SS effector protein as a target point is the most powerful, and a new direction is provided for the treatment of clinical drug-resistant strains.

Description

Design of polypeptide specifically binding to immune protein of pseudomonas aeruginosa hexa-type secretion system and verification of antibacterial activity of polypeptide

Technical Field

The invention relates to design of a polypeptide specifically binding to pseudomonas aeruginosa hexa-type secretory system immune protein (TpLEi) and verification of antibacterial activity of the polypeptide, in particular to a polypeptide which is designed based on a protein three-dimensional structure and targets the interaction of bacterial hexa-type secretory system effector protein-immune protein (TpLE-TpLEi), and the polypeptide is found to have antibacterial activity and has an anti-infective drug development prospect.

Background

Infectious diseases caused by pathogenic bacteria remain a significant threat to global public health since the twentieth century. The abuse of antibiotics has increasingly intensified the emergence of multi-and pan-resistant strains, and in particular, in recent years, the discovery of the resistance gene MCR-1 for polymyxin has been found, and the control of more pathogenic bacteria has led to an unprecedented challenge. Pseudomonas aeruginosa is the most typical representative of clinical drug-resistant bacteria, widely exists in nature, can be transmitted through ways such as environmental pollution, cross infection, endogenous infection and iatrogenic infection, and is an important nosocomial infectious bacterium causing pneumonia and surgical infection, particularly for patients with low immune function and ICU patients. In addition, P.aeruginosa is also a temptation for respiratory infections and chronic obstructive patients with cystic fibrosis. Currently, pseudomonas aeruginosa becomes an important conditional pathogen for nosocomial infection, accounts for about 11.1-24.5% of nosocomial infection cases in China, and occupies 2-3 th place. Whereas, on a global scale, P.aeruginosa infection has jumped to the first. The vast majority of these infections are caused by Multidrug (MDR) and pan-resistant (XDR) Pseudomonas aeruginosa, which has developed resistance to the last line of defense of antibiotics, polymyxin. Therefore, the design and development of new anti-infective drugs are urgently needed.

Currently, a number of new antibiotic alternatives are being developed, for example, Kenneth Shatzkes and colleagues use predatory bacteria to kill pneumococci in a mouse animal model. Phage therapy is also of great interest due to its high specificity, and phages directed against pseudomonas aeruginosa have been identified and used. In addition, antimicrobial peptides (AMPs) and metal ions are also under development. The most common methods of treating pseudomonas aeruginosa infections are antibiotic and phage therapy. However, the emergence of phage antibodies and drug resistance, among other things, exacerbate the failure of these therapeutic approaches. Anti-virulence strategies (anti-virus strands) are a new approach to anti-infective drug development that has been recently proposed. By interfering with the bacterial secretion system, inhibiting bacterial virulence can relieve pathogenic bacteria from "armed" to lose virulence without directly killing the bacteria. Because the bacteriostatic strategy does not directly endanger the survival of bacteria, the bacteriostatic strategy has smaller selective pressure, can delay the occurrence of drug-resistant mutation and causes great attention in international related fields.

The bacterial secretion system is a highly specialized biomacromolecule complex spanning multiple bacterial envelopes that can assist bacteria in delivering virulence effector proteins to a host cell or to a heterobacterium. A7-secretion system has now been found in bacteria (T1SS-T7 SS). The bacterial VI secretion system (T6SS) was the only recently discovered and described bacterial secretion system, widely implicated in pathogenicityIn gram-negative bacteria, the interaction between bacteria and a host is mediated by secreting effector toxin proteins, and the interaction plays an important role in the survival and pathogenesis of the bacteria. T6SS generally comprises a core module of 13-25 proteins or several accessory modules, which are functionally classified as structural, effector, regulatory and chaperone proteins. The effector protein, as an end product of T6SS, directly participates in competition between bacteria and pathogenicity of host. Therefore, T6SS and its effector proteins are ideal targets for drug design. Over the last few years, effector proteins of the T6SS secretory system have been identified in succession. Originally discovered to be secreted extracellularly are the structural components VgrG and Hcp of T6SS, which initiate infection by cross-linking actin in eukaryotic hosts. Subsequently, effector proteins that degrade bacterial cell walls (Tae/Tge), effector proteins that degrade cell membranes (Tle), effector proteins that degrade nucleic acids, and the like have been reported in succession. Interestingly, to protect autologous cells from poisoning by these effector proteins, T6SS⁺Bacteria simultaneously express homologous immunity proteins or antitoxin proteins (E-ipair) corresponding to various effector molecules to neutralize the effects of toxin effector proteins, which allows bacteria with T6SS to remain dominant in competition with other bacteria. Researches show that the virulence effector protein TpLE of the secretion system of the type VI pseudomonas aeruginosa directly participates in competition among bacterial species and autophagy of a host ER device as a cross-border virulence factor, and neutralizes the toxicity of the TpLE by expressing immune protein (antitoxic protein) TpEi so as to prevent suicide behavior.

Disclosure of Invention

The invention aims to design a polypeptide which is specifically combined with the hexa-type secretory system immune protein TpLEi and has antibacterial activity.

The invention firstly protects a polypeptide which is (a1), (a2), (a3), (a4), (a5), (a6), (a7) or (a 8):

(a1) polypeptide shown in sequence 3 of the sequence table;

(a2) a polypeptide derived therefrom, which is obtained by substituting and/or deleting and/or adding (a1) 1 to 5 amino acid residues and has the same function as the polypeptide;

(a3) polypeptide shown in sequence 4 of the sequence table;

(a4) a polypeptide obtained by attaching a tag to the N-terminus or/and the C-terminus of (a1) or (a 3);

(a5) a polypeptide obtained by linking a signal peptide to the N-terminus of (a1) or (a 3);

(a6) 1 st to 57 th amino acid residues of a sequence 11 in a sequence table;

(a7) a polypeptide obtained by connecting a signal peptide to the N-terminal of (a1) or (a3) and connecting a tag to the C-terminal;

(a8) polypeptide shown in sequence 11 of the sequence table.

The gene encoding the polypeptide also belongs to the protection scope of the invention.

The gene is (b1), (b2), (b3), (b4), (b5) or (b 6):

(b1) the coding region is shown as 70 th-171 th nucleotide of the sequence 10 in the sequence table;

(b2) the coding region is shown as 70 th-159 th nucleotides in the sequence 10 of the sequence table;

(b3) the coding region is shown as the DNA molecule shown by the 1 st to 171 th nucleotides of the sequence 10 in the sequence table;

(b4) the coding region is a DNA molecule shown as a sequence 10 in a sequence table;

(b5) a DNA molecule which hybridizes under stringent conditions with the DNA sequence defined in (b1) or (b2) or (b3) or (b4) and encodes the polypeptide;

(b6) a DNA molecule which has more than 90% of homology with the DNA sequence defined by (b1) or (b2) or (b3) or (b4) and encodes the polypeptide.

The recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain containing the gene belong to the protection scope of the invention.

The invention also protects the application of the polypeptide, which is (c1), (c2), (c3), (c4), (c5) or (c 6):

(c1) binding to an immunity protein in a bacterial type vi secretion system;

(c2) preparing a product for binding an immunity protein in a bacterial type vi secretion system;

(c3) enriching immune proteins in a bacterial type VI secretion system;

(c4) preparing a product for enriching an immune protein in a bacterial type VI secretion system;

(c5) detecting an immunity protein in a bacterial type VI secretion system;

(c6) preparing a product for detecting immune protein in a bacterial VI type secretion system.

The invention also protects the application of the polypeptide, which is (d1) or (d 2):

(d1) competitively binds to the immunity protein with the toxin protein in the bacterial type vi secretion system;

(d2) a product is prepared for competitive binding of the immunity protein to the toxin protein in the bacterial type vi secretion system.

The invention also protects the application of the polypeptide, which is (f1) or (f 2):

(f1) relieving the inhibition of the bacterial VI secretion system by immune protein to bacterial death induced by toxin protein;

(f1) preparing a product for relieving the inhibition of bacterial death induced by toxin proteins by immune proteins in a bacterial type VI secretion system.

The invention also protects the application of the polypeptide, which is (g1) or (g 2):

(g1) promoting the death of bacteria having a bacterial type vi secretion system;

(g1) preparing a product for promoting the death of bacteria having a bacterial type VI secretion system.

The bacterium having a bacterial type vi secretion system may be specifically pseudomonas aeruginosa.

Any one of the above toxin proteins is a TplE protein, specifically (h1), (h2), (h3), (h4), (h5) or (h 6):

(h1) a protein consisting of an amino acid sequence shown in a sequence 1 in a sequence table;

(h2) a protein which is derived from the sequence 1 and has the same function by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of the sequence 1;

(h3) a protein derived from pseudomonas aeruginosa and having more than 90% identity with the protein shown in the sequence 1;

(h4) a polypeptide obtained by attaching a tag to the N-terminus or/and the C-terminus of (h 1);

(h5) a polypeptide obtained by linking a signal peptide to the N-terminus of (h 1);

(h6) protein consisting of an amino acid sequence shown as a sequence 7 in a sequence table.

Any one of the above immune proteins is a tpliei protein, specifically the following (j1), (j2), (j3), (j4), (j5) or (j 6):

(j1) protein consisting of 26 th to 380 th amino acid residues in a sequence 2 in a sequence table;

(j2) a protein consisting of an amino acid sequence shown in a sequence 2 in a sequence table;

(j3) a protein derived from (j1) or (j2) by substitution and/or deletion and/or addition of one or more amino acid residues and having the same function;

(j4) a protein derived from pseudomonas aeruginosa and having more than 90% identity to (j1) or (j 2);

(j5) a protein obtained by attaching a tag to the N-terminus or/and the C-terminus of (j1) or (j 2);

(j6) and (j1) or (j2) to the N-terminus of the protein.

With the gradual increase of the drug resistance of bacteria, the development of novel anti-infective drugs is urgent. The type six secretion system (T6SS) is involved in prokaryotic and eukaryotic cell infections by secreting virulence proteins and plays an important role in ecosystem balance and human health. The effector protein TplE of the secretion system of pseudomonas aeruginosa type vi triggers its death by targeting the competitor's bacterial cell membrane, while itself neutralizes TplE toxicity by expressing the antitoxic protein TplEi, thereby preventing suicide.

The functional short peptide provided by the invention can be competitively combined with an effector protein (TpLE protein) to an immune protein (TpLE protein), so that the TpE-TpLE interaction is damaged, the cell membrane of effector protein cracked bacteria is promoted to cause the self-death of the bacteria, the novel antibacterial strategy which takes T6SS effector protein as a target point is the most powerful, the most direct drug screening platform and later-stage drug application and development are provided for designing novel anti-infective drugs, and a strong scientific and technological support is provided for the treatment of clinical drug-resistant strains.

Drawings

FIG. 1 shows peptide A and His₆-affinity assay results for TplEi.

FIG. 2 shows peptide B and His₆-affinity assay results for TplEi.

FIG. 3 shows the results of example 3.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

The TplE protein is an effector protein, also known as a toxin protein. The TpLE protein derived from pseudomonas aeruginosa is shown as a sequence 1 in a sequence table. The TpLEi protein is an immunity protein, also known as an antitoxin protein. The Pseudomonas aeruginosa-derived TpLEi protein is shown in a sequence 2 in a sequence table, wherein a signal peptide is from 1 to 25, and a mature peptide is from 26 to 380.

petDUET-1 vector: novagen Inc. Escherichia coli Rosetta (DE 3): novagen Inc. pET-28a (+) vector: novagen Inc. pET-26b (+) vector: novagen Inc. Escherichia coli BL 21: novagen Inc.

Example 1 discovery of functional short peptides

1. A petDUET-1 vector is used as a starting plasmid to construct a recombinant plasmid. In the recombinant plasmid, the first open reading frame expresses that the N end has His₆The tagged tpliei mature peptide, the second open reading frame expresses the TplE protein.

2. And (3) introducing the recombinant plasmid constructed in the step (1) into escherichia coli Rosetta (DE3) to obtain a recombinant bacterium.

3. Culturing the recombinant bacteria obtained in the step 2, carrying out IPTG induction in the culture process, and then sequentially carrying out the following steps: and (3) crushing the thalli, taking the supernatant, purifying by using a nickel column, performing ion exchange chromatography, and performing Superose 6 molecular sieve chromatography to obtain a TpLE-TpLEi compound.

4. And (3) adding subtilisin into the TpLE-TpLEi compound obtained in the step (3) for in-situ enzymolysis, digestion and crystallization, and then obtaining crystals and carrying out structure analysis.

5. And preparing the polypeptide according to the result of the structure analysis, and performing functional verification to finally find two functional short peptides which are named as A peptide and B peptide respectively.

Amino acid sequence of A peptide (sequence 3 of sequence table): DDLFASIGALWTWAWRGPKARQELLKAEQVEVDD are provided.

Amino acid sequence of B peptide (sequence 4 of sequence table): DDLFASIGALWTWAWRGPKARQELLKAEQ are provided.

Example 2 affinity of functional short peptides for TpLEi protein

And (3) carrying out an in vitro binding experiment on the functional short peptide and the TpLEi protein to determine the affinity of the functional short peptide. The high affinity of functional short peptides is the first step for the development of novel anti-infective drugs.

Firstly, preparing functional short peptide

Artificially synthesizing A peptide.

Artificially synthesizing the B peptide.

Secondly, preparation of TpLEi protein

1. Inserting a double-stranded DNA molecule shown by 64 th-1131 th nucleotides of a sequence 5 in a sequence table between NdeI and XhoI enzyme cutting sites of a pET-28a (+) vector to obtain a recombinant plasmid. Through sequencing verification, the recombinant plasmid has an open reading frame shown as a sequence 5 in a sequence table, and the expression N end has His₆The tag TplEi mature peptide.

2. And (3) introducing the recombinant plasmid obtained in the step (1) into escherichia coli Rosetta (DE3) to obtain a recombinant bacterium.

3. Inoculating the recombinant strain obtained in step 2 into liquid LB culture medium containing 50mg/L kanamycin and 50mg/L chloramphenicol, and performing shaking culture at 37 ℃ and 180rpm until OD is reached_600nmThe value is about 1.2.

4. After completion of step 3, the whole culture system was left in ice bath for half an hour, and then IPTG was added so that the concentration thereof in the system was 0.5mmol/L, followed by shaking culture at 180rpm at 18 ℃ for 18 hours.

5. After the step 4 is completed, centrifuging at 4000rpm for 15min, collecting thalli, suspending with lysate, performing ultrasonication on ice (power 600W, stopping 5s per 5s, 99 times), then centrifuging at 15000rpm for 30min, collecting supernatant, filtering the supernatant with 0.45 micron filter membrane, and collecting filtrate.

The lysate contains 2M NaCl, 12mM imidazole, 0.1% β -mercaptoethanol, 1mM PMSF, and the balance Tris-HCl buffer solution with pH7.5 and 50 mM.

6. Purification with Ni column

3ml nickel ion affinity chromatography column: QIAGEN.

The purification process comprises the steps of ① equilibrating the column with 10 column volumes of equilibration solution, ② loading the filtrate obtained in

step

5, ③ washing the column with 10 column volumes of washing solution to remove foreign proteins, ④ eluting the column with 15ml of eluent, and collecting the post-column solution.

Balance liquid: Tris-HCl buffer containing 2M NaCl, 12mM imidazole, the balance pH7.5, 50 mM.

Washing solution (ph 7.5): containing 150mM NaCl, 50mM Tris, 12mM imidazole, and the balance water.

The eluent (pH7.5) contains 75mM NaCl, 50mM Tris, 250mM imidazole, 0.1% β -mercaptoethanol and the balance water.

Filtering the solution after passing through the column by a 0.45-micron filter membrane, and collecting filtrate.

7. Purification by cation exchange chromatography

AKTA explore System.

5ml Hitrap SP HP column: GE HEALTHCARE are provided.

The purification process comprises the steps of balancing the column by ① buffer A with 5 column volumes, loading ② the filtrate obtained in the step 6, carrying out linear gradient elution by ③ buffer A and buffer B, monitoring the ultraviolet value at 280nm in real time, and collecting the solution after passing the column corresponding to the elution peak (only one obvious elution peak appears in the whole elution process).

buffer A (pH 7.5): 50mM Tris, 75mM NaCl, 1mM DTT, and the balance water.

buffer B (pH 7.5): containing 50mM Tris, 1M NaCl, 1mM DTT, and the balance being water.

And (3) carrying out ultrafiltration concentration on the solution after passing through the column by adopting an ultrafiltration concentration tube with the molecular weight cutoff of 10KD to obtain a protein concentrated solution.

8. Purifying by molecular sieve chromatography

24ml Superose 6 column chromatography: GE HEALTHCARE are provided.

The purification process comprises the steps of ① balancing columns by using molecular sieve buffer solution with the volume of 1 column, ② loading the protein concentrated solution obtained in the

step

7, ③ eluting by using the molecular sieve buffer solution, monitoring the ultraviolet value at 280nm in real time, and collecting the solution after passing the column corresponding to an elution peak (only one obvious elution peak appears in the whole elution process).

Molecular sieve buffer (ph 7.0): it contains 150mM NaCl, 20mM Tris and water for the rest.

Ultrafiltering and concentrating the solution after passing through the column with ultrafiltration concentration tube with cut-off molecular weight of 10KD to obtain protein concentrated solution containing His at N-terminal₆A solution of the tagged TpLEi mature peptide, designated His₆-TpLEi solution. The N terminal has His₆His for tagged TpLEi mature peptide₆-TplEi.

His₆In TpLEi solution, His₆The concentration of TpLEi was 10mg/ml (in total protein concentration).

Triple, affinity assay

An in vitro ITC experiment was performed using a MicroCal iTC200 microcalorimetric isothermal titration calorimeter. A solution of peptide A was prepared at a concentration of 1-2 mM. A solution of peptide B was prepared at a concentration of 1-2 mM. Preparation of His₆-dilutions of TpLEi solution with protein concentration of 0.03-0.04 mM. The solvents used to prepare each solution and dilution were as follows: 20mM Tris-HCl, 100mM NaCl, balance water, pH 8.0. Continuous injection was performed using 18 needles, 2 microliters per needle. At 120 second intervals, the data was finally processed using orgin.

Peptide A and His₆The results of the affinity assay for TpLEi are shown in FIG. 1. kd 125 nM.

B peptide and His₆The results of the affinity assay for TpLEi are shown in FIG. 2. kd 478 nM.

Example 3 initiation of bacterial autodeath by activation of TpLE protein by functional short peptides

Construction of recombinant plasmid

1. The double-stranded DNA molecule shown in sequence 6 of the sequence table was inserted between the Nco I and Xho I cleavage sites of pET-26b (+) vector to obtain recombinant plasmid pET26 b-TpLE. The recombinant plasmid pET26b-TpLE has an open reading frame shown in a sequence 6 of a sequence table through sequencing verification. The DNA molecule shown in the sequence 6 of the sequence table codes the protein shown in the sequence 7 of the sequence table. In the sequence 7 of the sequence table, the 1 st to 23 th amino acid residues form a signal peptide (promoting the secretion of the protein to the periplasmic space), and the 24 th to 592 th amino acid residues form a TpLE protein.

2. The double-stranded DNA molecule shown in sequence 8 of the sequence table was inserted between the Nco I and Xho I cleavage sites of pET-26b (+) vector to obtain recombinant plasmid pET26 b-TpLE-TpLEi. The recombinant plasmid pET26b-TpLE-TpLEi has an open reading frame shown in a sequence 8 of a sequence table through sequencing verification. The DNA molecule shown in the sequence 8 of the sequence table codes the protein shown in the sequence 9 of the sequence table. In the sequence 9 of the sequence table, the amino acid residues at the 1 st to the 23 th positions form a signal peptide (promoting the secretion of the protein to the periplasmic space), the amino acid residues at the 24 th to the 592 th positions form a TpLE protein, and the amino acid residues at the 593 rd and 972 th positions form a TpLEi protein.

3. Double-stranded DNA molecules shown by 1 st to 171 th nucleotides in a sequence 10 of a sequence table are inserted between Nco I and Xho I enzyme cutting sites of a pET-26b (+) vector to obtain a recombinant plasmid pET26 b-A. The recombinant plasmid pET26b-A has an open reading frame shown in a sequence 10 of a sequence table through sequencing verification. The DNA molecule shown in the sequence 10 of the sequence table codes the protein shown in the sequence 11 of the sequence table. In the sequence 11 of the sequence table, the 1 st to 23 th amino acid residues form a signal peptide (promoting the secretion of protein to periplasmic space), and the 24 th to 57 th amino acid residues form an A peptide.

4. Double-stranded DNA molecules shown by 1 st to 171 th nucleotides of a sequence 12 in a sequence table are inserted between Nco I and Xho I enzyme cutting sites of a pET-26b (+) vector to obtain a recombinant plasmid pET26 b-K100E. Sequencing verification shows that the recombinant plasmid pET26b-K100E has an open reading frame shown in a sequence 12 of a sequence table.

5. The DNA molecule shown by 1 st to 171 th nucleotides in the sequence 13 of the sequence table is inserted between the Nco I and Xho I enzyme cutting sites of pET-26b (+) vector to obtain recombinant plasmid pET26 b-W92A. Sequencing verification shows that the recombinant plasmid pET26b-W92A has an open reading frame shown in a sequence 13 of a sequence table.

Second, bacterial culture test

A first group: introducing a pET-26b (+) vector into escherichia coli BL21, then selecting a recombinant bacterium for monoclonal inoculation to 4ml of liquid LB culture medium, carrying out shaking culture at 37 ℃ and 220rpm for 10 hours, then sampling a bacterium liquid, carrying out 10-fold gradient dilution by using the liquid LB culture medium, then dropwise adding 2 microliters to an LB culture medium plate, carrying out standing culture at 37 ℃ for 24 hours, and taking a picture.

Second group: introducing the recombinant plasmid pET26b-TpLE into escherichia coli BL21, then selecting recombinant bacteria for single cloning, inoculating the recombinant bacteria into 4ml of liquid LB culture medium, carrying out shaking culture at 37 ℃ and 220rpm for 10 hours, then sampling bacterial liquid, carrying out 10-fold gradient dilution by adopting the liquid LB culture medium, then dropwise adding 2 microliters to an LB culture medium plate, carrying out standing culture at 37 ℃ for 24 hours, and taking a picture.

Third group: introducing the recombinant plasmid pET26b-TpLE-TpLEi into escherichia coli BL21, then selecting recombinant bacteria for single cloning and inoculating to 4ml of liquid LB culture medium, carrying out shaking culture at 37 ℃ and 220rpm for 10 hours, then sampling bacterial liquid and carrying out 10-fold gradient dilution by adopting the liquid LB culture medium, then dropwise adding 2 microliter to an LB culture medium plate, standing and culturing at 37 ℃ for 24 hours, and taking pictures.

And a fourth group: introducing the recombinant plasmid pET26b-TplE-TplEi and pET-26b (+) vector into Escherichia coli BL21, then selecting recombinant bacteria to be singly inoculated to 4ml of liquid LB culture medium, carrying out shaking culture at 37 ℃ and 220rpm for 10 hours, then sampling bacterial liquid, carrying out 10-fold gradient dilution by adopting the liquid LB culture medium, then dropwise adding 2 microliter to an LB culture medium plate, carrying out standing culture at 37 ℃ for 24 hours, and taking pictures.

And a fifth group: the recombinant plasmid pET26b-TpLE-TpLEi and the recombinant plasmid pET26b-A are co-introduced into escherichia coli BL21, then the recombinant bacterium is selected to be singly inoculated into 4ml of liquid LB culture medium, shaking culture is carried out at 37 ℃ and 220rpm for 10 hours, then the bacterium liquid is sampled and is subjected to 10-fold gradient dilution by adopting the liquid LB culture medium, then 2 microlitres is dripped onto an LB culture medium plate, standing culture is carried out at 37 ℃ for 24 hours, and photographing is carried out.

A sixth group: the recombinant plasmid pET26b-TpLE-TpLEi and the recombinant plasmid pET26b-W92A are co-introduced into escherichia coli BL21, then the recombinant bacterium is selected to be monoclonal and inoculated into 4ml of liquid LB culture medium, shaking culture is carried out at 37 ℃ and 220rpm for 10 hours, then the bacterium liquid is sampled and is subjected to 10-fold gradient dilution by adopting the liquid LB culture medium, then 2 microlitres is dripped onto an LB culture medium plate, standing culture is carried out at 37 ℃ for 24 hours, and pictures are taken.

A seventh group: the recombinant plasmid pET26b-TpLE-TpLEi and the recombinant plasmid pET26b-K100E are co-introduced into escherichia coli BL21, then the recombinant bacterium is selected to be monoclonal and inoculated to 4ml of liquid LB culture medium, shaking culture is carried out at 37 ℃ and 220rpm for 10 hours, then the bacterium liquid is sampled and is subjected to 10-fold gradient dilution by adopting the liquid LB culture medium, then 2 microlitres is dripped onto an LB culture medium plate, standing culture is carried out at 37 ℃ for 24 hours, and photographing is carried out.

And an eighth group: the pET-26b (+) vector and the recombinant plasmid pET26b-A are co-introduced into escherichia coli BL21, then the recombinant bacterium is selected to be monoclonal inoculated to 4ml of liquid LB culture medium, the liquid LB culture medium is oscillated and cultured at 37 ℃ and 220rpm for 10 hours, then the bacterium liquid is sampled and is subjected to 10-fold gradient dilution by adopting the liquid LB culture medium, then 2 microlitres is dripped onto an LB culture medium plate, the liquid is kept still and cultured at 37 ℃ for 24 hours, and pictures are taken.

The results are shown in FIG. 3. In FIG. 3, each group is, from left to right, a bacterial liquid, a 10-fold dilution of the bacterial liquid, a 100-fold dilution of the bacterial liquid, and a 10-fold dilution of the bacterial liquid ³10 of dilution liquid and bacterial liquid ⁴10 of dilution and bacterial liquid⁵Double dilution. The number of surviving recombinant bacteria was significantly reduced in the second group compared to the first group, i.e. the TplE protein induced bacterial death. The number of surviving recombinant bacteria of the third group was significantly increased compared to the second group, i.e., the TplE protein had an inhibitory effect on the TplE protein-induced bacterial death. Compared with the fourth group, the survival number of the fifth group of recombinant bacteria is obviously reduced, namely the A peptide is combined with TpLE protein competitively, so that the inhibition effect of the TpLE protein on the TpLE protein-induced bacterial death is relieved, the TpLE protein is released to play a toxic role, and the bacteria die. The survival number of the recombinant bacteria in the sixth and seventh groups was significantly increased compared to the fifth group, i.e., after point mutation of the A peptideThe corresponding effect is remarkably reduced. The results in the eighth group show that there is essentially no lethal effect of the A peptide itself on the bacteria.

SEQUENCE LISTING

<110> institute of pathogenic biology of Chinese academy of medical sciences

<120> design of polypeptide specifically binding to immune protein of P.aeruginosa hexatype secretory system and verification of antibacterial activity thereof

<130>GNCYX171704

<160>13

<170>PatentIn version 3.5

<210>1

<211>569

<212>PRT

<213>P.Aeruginosa

<400>1

Met Ser Ser Glu Pro Leu Glu Pro Asn Gln Asp Val Ile Ile Pro Arg

1 5 10 15

Ser Arg Asp Ser Leu Gly Arg Pro Val Tyr Lys Ala Gln Leu Thr Arg

20 25 30

Thr Asp Asn Gln Ser Glu Lys Val Ala Leu Ile Arg Gln Thr Ala Pro

35 40 45

Leu Pro Val Ile Phe Ile Pro Gly Ile Met Gly Thr Asn Leu Arg Asn

50 55 60

Lys Ala Asp Lys Ser Glu Val Trp Arg Pro Pro Asn Gly Leu Trp Pro

65 70 75 80

Met AspAsp Leu Phe Ala Ser Ile Gly Ala Leu Trp Thr Trp Ala Trp

85 90 95

Arg Gly Pro Lys Ala Arg Gln Glu Leu Leu Lys Ala Glu Gln Val Glu

100 105 110

Val Asp Asp Gln Gly Thr Ile Asp Val Gly Gln Ser Gly Leu Ser Glu

115 120 125

Glu Ala Ala Arg Leu Arg Gly Trp Gly Lys Val Met Arg Ser Ala Tyr

130 135 140

Asn Pro Val Met Gly Leu Met Glu Arg Arg Leu Asp Asn Ile Val Ser

145 150 155 160

Arg Arg Glu Leu Gln Ala Trp Trp Asn Asp Glu Ala Leu Ser Pro Pro

165 170 175

Gly Asp Gln Gly Glu Glu Gln Gly Lys Val Gly Pro Ile Asp Glu Glu

180 185 190

Glu Leu Leu Arg Ala Ser Arg Tyr Gln Phe Asp Val Trp Cys Ala Gly

195 200 205

Tyr Asn Trp Leu Gln Ser Asn Arg Gln Ser Ala Leu Asp Val Arg Asp

210 215 220

Tyr Ile Glu Asn Thr Val Leu Pro Phe Tyr Gln Lys Glu Cys Gly Leu

225 230 235 240

Asp Pro Glu Gln Met Arg Arg Met Lys Val Ile Leu Val Thr His Ser

245 250 255

Met Gly Gly Leu Val Ala Arg Ala Leu Thr Gln Leu His Gly Tyr Glu

260 265 270

Arg Val Leu Gly Val Val His Gly Val Gln Pro Ala Thr Gly Ser Ser

275 280 285

Thr Ile Tyr His His Met Arg Cys Gly Tyr Glu Gly Ile Ala Gln Val

290 295 300

Val Leu Gly Arg Asn Ala Gly Glu Val Thr Ala Ile Val Ala Asn Ser

305 310 315 320

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

325 330 335

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

340 345 350

Asp Gly Lys Pro Arg Ala Tyr Pro Gln Asn Gln Asp Pro Tyr Glu Glu

355 360 365

Ile Tyr Lys Asn Thr Thr Trp Tyr Gly Leu Val Pro Glu Gln Asn Ser

370 375 380

Gln Tyr Leu Asp Met Ser Asp Lys Lys Glu Gly Leu Arg Val Gly Pro

385 390 395 400

Arg Asp Asn Phe Glu Asp Leu Ile Asp Ser Ile Ala Asn Phe His Gly

405 410 415

Glu Leu Ser Ala Ala Gly Tyr His Ser Glu Thr Tyr Ala His Tyr Gly

420 425 430

Ala Asp Asp Ser Arg His Ser Trp Arg Asp Leu Ile Trp Lys Gly Asp

435 440 445

Pro Thr Pro Leu Glu Thr Pro Gly Ala Thr Leu Asn Asp Asp Glu Asn

450 455 460

Gly Thr Tyr Asn Ser Trp Phe Arg Arg Gly Leu Pro Thr Ile Val Gln

465 470 475 480

Gly Pro Leu Glu Thr Gly Asn Pro Leu Asp Ala Ser Gly Ser Gly Gly

485 490 495

Asp Glu Thr Val Pro Thr Asp Ser Gly Gln Ala Pro Ala Leu Ala Gly

500 505 510

Val Lys Ala Ser Phe Arg His Gly Ser Lys Gly Lys Gly Gln Ala Asn

515 520 525

Thr Lys Arg Gly Tyr Glu His Gln Glu Ser Tyr Asn Asp Ala Arg Ala

530 535 540

Gln Trp Ala Ala Leu Tyr Gly Val Ile Lys Ile Thr Gln Leu Ala Asp

545 550 555 560

Trp His Pro Asn Asp Lys Gly Gly Thr

565

<210>2

<211>380

<212>PRT

<213>P.Aeruginosa

<400>2

Met Met His Pro Arg Arg Leu Leu Ala Val Leu Gly Val Val Val Leu

1 5 10 15

Thr Ser Met Thr Ser Cys Thr Ser Phe Ser Ser Ser Arg Ser Ser Ser

20 25 30

Met Asp Lys Thr Gly Trp Ile Thr His Cys Phe Gly Arg Phe Leu Ile

35 40 45

Asp Leu Pro Pro Asp Ala Val Ile Asn Ala Gly Tyr Tyr Leu Trp Gly

50 55 60

Asp Arg Ile Glu Tyr Leu Asp Asp Lys Pro Thr Glu Leu Ala Ala Arg

65 70 75 80

Val Asp Arg Leu Glu Gln Glu Trp Arg Thr Gln Arg His Lys Ser Lys

85 90 95

Gly Asn Met Phe Leu Arg Lys Ile Asp Phe Gly Asn Glu Ser Val Gly

100 105 110

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

115 120 125

Thr Tyr Val Thr SerLys Pro Thr Trp His Val Tyr Arg Trp Lys Gly

130 135 140

Lys Val Ser Val Asp Arg Glu Gln His Ala Val Glu Ile Ser Arg Ala

145 150 155 160

Leu Ala Arg Asn Leu Arg Ser Arg Ala Pro Lys Glu Ile Pro Ser Glu

165 170 175

Pro Gly Phe Cys Ile Asp His Ala Tyr Ile Ala Gly Asp Ser Phe Gln

180 185 190

Val Glu Arg Phe Gly Val Gly Val Thr Phe Pro Glu His Pro Gly Ala

195 200 205

Arg Phe Glu Phe Arg Ser Ser Thr Gly Ala Glu Leu Asn Ser Leu Leu

210 215 220

Glu Arg Val Asp Gly Phe Val Gln Asn Met Leu Ser Thr Phe Ala Gly

225 230 235 240

Met Glu Thr Leu Arg Lys Gly Lys His Pro Val Gly Ser Leu Pro Gly

245 250 255

Glu Glu Tyr Leu Val Ala Gly Ser Asp Lys Gly Gln Arg Gly Tyr Thr

260 265 270

Phe Met Trp Glu Val Gln Gly Lys Glu Glu Ser Leu Thr Glu Pro Asn

275 280 285

Leu Thr Ala Gly Leu Ala ValLeu Glu Arg Ser Asn Glu Asn Gly Lys

290 295 300

Pro Pro Pro Pro Ala Phe Lys Ser Asp Lys Glu Ala Leu Glu Leu Trp

305 310 315 320

Asp Thr Ile Val Asp Ser Ile Arg Val Arg Pro Thr Ser Ser Ser Pro

325 330 335

Arg Gly Gly Asn Ala Gly Pro Ser Pro Ala Pro Lys Pro Ala Thr Pro

340 345 350

Gly Gly Gln Thr Leu Gly Asp His Tyr Val Tyr Glu Glu Phe Leu Ser

355 360 365

Ser Leu Lys Pro Lys Asp Ser Trp Leu Asp Asp Leu

370 375 380

<210>3

<211>34

<212>PRT

<213>Artificial sequence

<400>3

Asp Asp Leu Phe Ala Ser Ile Gly Ala Leu Trp Thr Trp Ala Trp Arg

1 5 10 15

Gly Pro Lys Ala Arg Gln Glu Leu Leu Lys Ala Glu Gln Val Glu Val

20 25 30

Asp Asp

<210>4

<211>29

<212>PRT

<213>Artificial sequence

<400>4

Asp Asp Leu Phe Ala Ser Ile Gly Ala Leu Trp Thr Trp Ala Trp Arg

1 5 10 15

Gly Pro Lys Ala Arg Gln Glu Leu Leu Lys Ala Glu Gln

20 25

<210>5

<211>1131

<212>DNA

<213>Artificial sequence

<400>5

atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60

atgtcttctt ctcggagctc ttccatggac aagaccggat ggattactca ttgctttggt 120

cgctttctga ttgatctgcc acctgacgcg gtaatcaatg ccggatacta cctatgggga 180

gacagaattg agtacttaga tgataaacct accgaactgg cagccagggt tgatcgtctt 240

gaacaagagt ggaggactca gagacataaa tccaaaggta acatgtttct tcgtaaaatt 300

gactttggca atgaatcagt aggtttattg tcttggtctt cagaggttgc ctctaaaacc 360

tatttattgg atacctatgt tacatctaag cctacgtggc atgtctatcg ttggaaaggg 420

aaagtttcgg tagacagaga gcaacatgct gtggagatat ctagggcttt agctcgaaat 480

ctacgttctc gcgcgccaaa agaaatcccc agcgagccgg gcttttgcat cgaccacgct 540

tatattgcag gggatagttt tcaagtggag aggtttggag tgggagtcac tttccctgag 600

caccccggcg cgcgctttga attccgctcc tccacgggag ctgagctaaa cagccttctt 660

gagcgagtcg acggcttcgt gcaaaacatg ctctccacgt ttgcaggtat ggaaacatta 720

agaaaaggaa agcatcctgt cgggagcctt cccggagagg agtatctggt tgcgggaagc 780

gataaggggc agcgcggcta taccttcatg tgggaggttc aaggcaagga ggaatcgcta 840

accgagccca atcttactgc gggactggcg gtactggaac gcagcaatga aaacggtaaa 900

ccaccgcccc ctgccttcaa atcggataaa gaagcattgg aactgtggga caccattgtc 960

gattcgattc gcgtgcgccc gacgtcttca tcgccacgtg gcggaaatgc cggtccttcc 1020

cctgctccga aacccgctac gcctggcggc caaacgctcg gtgatcacta tgtctatgag 1080

gaattcctct ccagcctgaa gccaaaagac agctggctgg acgacctata g 1131

<210>6

<211>1779

<212>DNA

<213>Artificial sequence

<400>6

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60

atggccatga tgagcagcga accgctcgag ccgaatcagg atgtgatcat ccctcgctct 120

cgcgacagtc tgggacggcc tgtctacaag gcgcagttga cccgtaccga caaccagagc 180

gaaaaggtgg cactgattcg gcagaccgct cccctgccgg tgatcttcat tccggggatc 240

atgggaacca acctgcgcaa caaggcagat aaaagcgagg tctggaggcc gccgaatggt 300

ctgtggccca tggatgacct gtttgcctcc atcggtgcgt tgtggacttg ggcctggagg 360

ggtccaaagg cccgtcagga gctcctgaag gctgagcaag tagaagtcga tgaccagggt 420

acgatcgatg tcggtcaatctggtttgtcc gaagaagctg cccgcctgcg cggctggggc 480

aaggtcatgc gcagtgcgta taacccggtc atgggattaa tggagaggcg actggataac 540

atcgtcagcc ggcgggaact ccaggcatgg tggaatgacg aagccctctc tcctcctgga 600

gatcagggtg aggagcaggg aaaggttggg ccgatcgatg aagaggaatt gctcagggcc 660

agccgatacc agttcgatgt ctggtgcgcc ggctataact ggctacagtc caaccggcag 720

tcggcgctgg atgtacgcga ctatatcgag aataccgtgt tgcctttcta ccaaaaggag 780

tgcggtctcg atcccgagca gatgagacgt atgaaggtca ttctggtcac ccattcgatg 840

ggagggctgg tggctcgcgc cctgacgcag ttgcacggct atgagcgcgt attgggggtg 900

gtgcatggcg tacaacctgc gacaggttcc tcgaccatct accatcatat gcgctgtggc 960

tatgaaggta tcgcacaggt cgtgctgggg cgaaatgccg gtgaagtgac agccattgtc 1020

gccaattcgg ccggcgcgct ggaattggcc cctagcgcgg aataccggga agggcgtccc 1080

tggctgtttc tatgcgacgc gcaggggcag gtgctcaagg atatcgatgg aaagcctcgg 1140

gcttatccgc agaatcagga cccttatgaa gagatataca agaacactac gtggtatgga 1200

ttggtgcctg agcaaaattc acaatatctg gacatgtcag acaaaaaaga aggtttaaga 1260

gttggccccc gtgataactt tgaagacttg atagatagca ttgctaattt tcatggtgaa 1320

ttatcagcag caggatatca ctcagagacc tatgcccatt acggagctga cgatagccgc 1380

catagttggc gcgacctgat ctggaagggt gaccctaccc ctctggagac ccccggtgcc 1440

acactcaacg acgatgagaa cggcacttac aacagttggt tccgccgggg cttgcccacc 1500

atagtgcagg gtccgctgga gacgggaaat cctctggacgcttccggtag cggaggtgac 1560

gaaacggtac cgaccgactc cggccaggcg cctgccctgg cgggcgtcaa ggccagcttt 1620

cggcatggca gcaagggcaa gggacaggcc aataccaaac ggggctatga gcaccaggag 1680

agttacaacg atgctcgtgc tcaatgggca gcgctatatg gggtgatcaa aatcacccag 1740

ttggcggatt ggcatcctaa tgacaaagga gggacatga 1779

<210>7

<211>592

<212>PRT

<213>Artificial sequence

<400>7

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Met Met Ser Ser Glu Pro Leu Glu Pro Asn

20 25 30

Gln Asp Val Ile Ile Pro Arg Ser Arg Asp Ser Leu Gly Arg Pro Val

35 40 45

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

50 55 60

Leu Ile Arg Gln Thr Ala Pro Leu Pro Val Ile Phe Ile Pro Gly Ile

65 70 75 80

Met Gly Thr Asn Leu Arg Asn Lys Ala Asp Lys Ser Glu Val Trp Arg

85 90 95

Pro Pro Asn Gly Leu Trp Pro Met Asp Asp Leu Phe Ala Ser Ile Gly

100 105 110

Ala Leu Trp Thr Trp Ala Trp Arg Gly Pro Lys Ala Arg Gln Glu Leu

115 120 125

Leu Lys Ala Glu Gln Val Glu Val Asp Asp Gln Gly Thr Ile Asp Val

130 135 140

Gly Gln Ser Gly Leu Ser Glu Glu Ala Ala Arg Leu Arg Gly Trp Gly

145 150 155 160

Lys Val Met Arg Ser Ala Tyr Asn Pro Val Met Gly Leu Met Glu Arg

165 170 175

Arg Leu Asp Asn Ile Val Ser Arg Arg Glu Leu Gln Ala Trp Trp Asn

180 185 190

Asp Glu Ala Leu Ser Pro Pro Gly Asp Gln Gly Glu Glu Gln Gly Lys

195 200 205

Val Gly Pro Ile Asp Glu Glu Glu Leu Leu Arg Ala Ser Arg Tyr Gln

210 215 220

Phe Asp Val Trp Cys Ala Gly Tyr Asn Trp Leu Gln Ser Asn Arg Gln

225 230 235 240

Ser Ala Leu Asp Val Arg Asp Tyr Ile Glu Asn Thr Val Leu Pro Phe

245 250 255

Tyr Gln Lys Glu Cys Gly Leu Asp Pro Glu Gln Met Arg Arg Met Lys

260 265 270

Val Ile Leu Val Thr His Ser Met Gly Gly Leu Val Ala Arg Ala Leu

275 280 285

Thr Gln Leu His Gly Tyr Glu Arg Val Leu Gly Val Val His Gly Val

290 295 300

Gln Pro Ala Thr Gly Ser Ser Thr Ile Tyr His His Met Arg Cys Gly

305 310 315 320

Tyr Glu Gly Ile Ala Gln Val Val Leu Gly Arg Asn Ala Gly Glu Val

325 330 335

Thr Ala Ile Val Ala Asn Ser Ala Gly Ala Leu Glu Leu Ala Pro Ser

340 345 350

Ala Glu Tyr Arg Glu Gly Arg Pro Trp Leu Phe Leu Cys Asp Ala Gln

355 360 365

Gly Gln Val Leu Lys Asp Ile Asp Gly Lys Pro Arg Ala Tyr Pro Gln

370 375 380

Asn Gln Asp Pro Tyr Glu Glu Ile Tyr Lys Asn Thr Thr Trp Tyr Gly

385 390 395 400

Leu Val Pro Glu Gln Asn Ser Gln Tyr Leu Asp Met Ser Asp Lys Lys

405 410 415

Glu Gly Leu Arg Val Gly Pro Arg Asp Asn Phe Glu Asp Leu Ile Asp

420 425 430

Ser Ile Ala Asn Phe His Gly Glu Leu Ser Ala Ala Gly Tyr His Ser

435 440 445

Glu Thr Tyr Ala His Tyr Gly Ala Asp Asp Ser Arg His Ser Trp Arg

450 455 460

Asp Leu Ile Trp Lys Gly Asp Pro Thr Pro Leu Glu Thr Pro Gly Ala

465 470 475 480

Thr Leu Asn Asp Asp Glu Asn Gly Thr Tyr Asn Ser Trp Phe Arg Arg

485 490 495

Gly Leu Pro Thr Ile Val Gln Gly Pro Leu Glu Thr Gly Asn Pro Leu

500 505 510

Asp Ala Ser Gly Ser Gly Gly Asp Glu Thr Val Pro Thr Asp Ser Gly

515 520 525

Gln Ala Pro Ala Leu Ala Gly Val Lys Ala Ser Phe Arg His Gly Ser

530 535 540

Lys Gly Lys Gly Gln Ala Asn Thr Lys Arg Gly Tyr Glu His Gln Glu

545 550 555 560

Ser Tyr Asn Asp Ala Arg Ala Gln Trp Ala Ala Leu Tyr Gly Val Ile

565 570 575

Lys Ile Thr Gln Leu Ala Asp Trp His Pro Asn Asp Lys Gly Gly Thr

580 585 590

<210>8

<211>2919

<212>DNA

<213>Artificial sequence

<400>8

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60

atggccatga tgagcagcga accgctcgag ccgaatcagg atgtgatcat ccctcgctct 120

cgcgacagtc tgggacggcc tgtctacaag gcgcagttga cccgtaccga caaccagagc 180

gaaaaggtgg cactgattcg gcagaccgct cccctgccgg tgatcttcat tccggggatc 240

atgggaacca acctgcgcaa caaggcagat aaaagcgagg tctggaggcc gccgaatggt 300

ctgtggccca tggatgacct gtttgcctcc atcggtgcgt tgtggacttg ggcctggagg 360

ggtccaaagg cccgtcagga gctcctgaag gctgagcaag tagaagtcga tgaccagggt 420

acgatcgatg tcggtcaatc tggtttgtcc gaagaagctg cccgcctgcg cggctggggc 480

aaggtcatgc gcagtgcgta taacccggtc atgggattaa tggagaggcg actggataac 540

atcgtcagcc ggcgggaact ccaggcatgg tggaatgacg aagccctctc tcctcctgga 600

gatcagggtg aggagcaggg aaaggttggg ccgatcgatg aagaggaatt gctcagggcc 660

agccgatacc agttcgatgt ctggtgcgcc ggctataact ggctacagtc caaccggcag 720

tcggcgctgg atgtacgcga ctatatcgag aataccgtgt tgcctttcta ccaaaaggag 780

tgcggtctcg atcccgagca gatgagacgt atgaaggtca ttctggtcac ccattcgatg 840

ggagggctgg tggctcgcgc cctgacgcag ttgcacggct atgagcgcgt attgggggtg 900

gtgcatggcg tacaacctgc gacaggttcc tcgaccatct accatcatat gcgctgtggc 960

tatgaaggta tcgcacaggt cgtgctgggg cgaaatgccg gtgaagtgac agccattgtc 1020

gccaattcgg ccggcgcgct ggaattggcc cctagcgcgg aataccggga agggcgtccc 1080

tggctgtttc tatgcgacgc gcaggggcag gtgctcaagg atatcgatgg aaagcctcgg 1140

gcttatccgc agaatcagga cccttatgaa gagatataca agaacactac gtggtatgga 1200

ttggtgcctg agcaaaattc acaatatctg gacatgtcag acaaaaaaga aggtttaaga 1260

gttggccccc gtgataactt tgaagacttg atagatagca ttgctaattt tcatggtgaa 1320

ttatcagcag caggatatca ctcagagacc tatgcccatt acggagctga cgatagccgc 1380

catagttggc gcgacctgat ctggaagggt gaccctaccc ctctggagac ccccggtgcc 1440

acactcaacg acgatgagaa cggcacttac aacagttggt tccgccgggg cttgcccacc 1500

atagtgcagg gtccgctgga gacgggaaat cctctggacg cttccggtag cggaggtgac 1560

gaaacggtac cgaccgactc cggccaggcg cctgccctgg cgggcgtcaa ggccagcttt 1620

cggcatggca gcaagggcaa gggacaggcc aataccaaac ggggctatga gcaccaggag 1680

agttacaacg atgctcgtgc tcaatgggca gcgctatatg gggtgatcaa aatcacccag 1740

ttggcggatt ggcatcctaa tgacaaagga gggacaatga tgcatccccg tcggttgctt 1800

gctgtacttg gagtggtggt cttgaccagc atgaccagtt gcacttcgtt ttcttcttct 1860

cggagctctt ccatggacaa gaccggatgg attactcatt gctttggtcg ctttctgatt 1920

gatctgccac ctgacgcggt aatcaatgcc ggatactacc tatggggaga cagaattgag 1980

tacttagatg ataaacctac cgaactggca gccagggttg atcgtcttga acaagagtgg 2040

aggactcaga gacataaatc caaaggtaac atgtttcttc gtaaaattga ctttggcaat 2100

gaatcagtag gtttattgtc ttggtcttca gaggttgcct ctaaaaccta tttattggat 2160

acctatgtta catctaagcc tacgtggcat gtctatcgtt ggaaagggaa agtttcggta 2220

gacagagagc aacatgctgt ggagatatct agggctttag ctcgaaatct acgttctcgc 2280

gcgccaaaag aaatccccag cgagccgggc ttttgcatcg accacgctta tattgcaggg 2340

gatagttttc aagtggagag gtttggagtg ggagtcactt tccctgagca ccccggcgcg 2400

cgctttgaat tccgctcctc cacgggagct gagctaaaca gccttcttga gcgagtcgac 2460

ggcttcgtgc aaaacatgct ctccacgttt gcaggtatgg aaacattaag aaaaggaaag 2520

catcctgtcg ggagccttcc cggagaggag tatctggttg cgggaagcga taaggggcag 2580

cgcggctata ccttcatgtg ggaggttcaa ggcaaggagg aatcgctaac cgagcccaat 2640

cttactgcgg gactggcggt actggaacgc agcaatgaaa acggtaaacc accgccccct 2700

gccttcaaat cggataaaga agcattggaa ctgtgggaca ccattgtcga ttcgattcgc 2760

gtgcgcccga cgtcttcatc gccacgtggc ggaaatgccg gtccttcccc tgctccgaaa 2820

cccgctacgc ctggcggcca aacgctcggt gatcactatg tctatgagga attcctctcc 2880

agcctgaagc caaaagacag ctggctggac gacctatag 2919

<210>9

<211>972

<212>PRT

<213>Artificial sequence

<400>9

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Met Met Ser Ser Glu Pro Leu Glu Pro Asn

20 25 30

Gln Asp Val Ile Ile Pro Arg Ser Arg Asp Ser Leu Gly Arg Pro Val

35 40 45

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

50 55 60

Leu Ile Arg Gln Thr Ala Pro Leu Pro Val Ile Phe Ile Pro Gly Ile

65 70 75 80

Met Gly Thr Asn Leu Arg Asn Lys Ala Asp Lys Ser Glu Val Trp Arg

85 90 95

Pro Pro Asn Gly Leu Trp Pro Met Asp Asp Leu Phe Ala Ser Ile Gly

100 105 110

Ala Leu Trp Thr Trp Ala Trp Arg Gly Pro Lys Ala Arg Gln Glu Leu

115 120 125

Leu Lys Ala Glu Gln Val Glu Val Asp Asp Gln Gly Thr Ile Asp Val

130 135 140

Gly Gln Ser Gly Leu Ser Glu Glu Ala Ala Arg Leu Arg Gly Trp Gly

145150 155 160

Lys Val Met Arg Ser Ala Tyr Asn Pro Val Met Gly Leu Met Glu Arg

165 170 175

Arg Leu Asp Asn Ile Val Ser Arg Arg Glu Leu Gln Ala Trp Trp Asn

180 185 190

Asp Glu Ala Leu Ser Pro Pro Gly Asp Gln Gly Glu Glu Gln Gly Lys

195 200 205

Val Gly Pro Ile Asp Glu Glu Glu Leu Leu Arg Ala Ser Arg Tyr Gln

210 215 220

Phe Asp Val Trp Cys Ala Gly Tyr Asn Trp Leu Gln Ser Asn Arg Gln

225 230 235 240

Ser Ala Leu Asp Val Arg Asp Tyr Ile Glu Asn Thr Val Leu Pro Phe

245 250 255

Tyr Gln Lys Glu Cys Gly Leu Asp Pro Glu Gln Met Arg Arg Met Lys

260 265 270

Val Ile Leu Val Thr His Ser Met Gly Gly Leu Val Ala Arg Ala Leu

275 280 285

Thr Gln Leu His Gly Tyr Glu Arg Val Leu Gly Val Val His Gly Val

290 295 300

Gln Pro Ala Thr Gly Ser Ser Thr Ile Tyr His His Met Arg Cys Gly

305310 315 320

Tyr Glu Gly Ile Ala Gln Val Val Leu Gly Arg Asn Ala Gly Glu Val

325 330 335

Thr Ala Ile Val Ala Asn Ser Ala Gly Ala Leu Glu Leu Ala Pro Ser

340 345 350

Ala Glu Tyr Arg Glu Gly Arg Pro Trp Leu Phe Leu Cys Asp Ala Gln

355 360 365

Gly Gln Val Leu Lys Asp Ile Asp Gly Lys Pro Arg Ala Tyr Pro Gln

370 375 380

Asn Gln Asp Pro Tyr Glu Glu Ile Tyr Lys Asn Thr Thr Trp Tyr Gly

385 390 395 400

Leu Val Pro Glu Gln Asn Ser Gln Tyr Leu Asp Met Ser Asp Lys Lys

405 410 415

Glu Gly Leu Arg Val Gly Pro Arg Asp Asn Phe Glu Asp Leu Ile Asp

420 425 430

Ser Ile Ala Asn Phe His Gly Glu Leu Ser Ala Ala Gly Tyr His Ser

435 440 445

Glu Thr Tyr Ala His Tyr Gly Ala Asp Asp Ser Arg His Ser Trp Arg

450 455 460

Asp Leu Ile Trp Lys Gly Asp Pro Thr Pro Leu Glu Thr Pro Gly Ala

465 470475 480

Thr Leu Asn Asp Asp Glu Asn Gly Thr Tyr Asn Ser Trp Phe Arg Arg

485 490 495

Gly Leu Pro Thr Ile Val Gln Gly Pro Leu Glu Thr Gly Asn Pro Leu

500 505 510

Asp Ala Ser Gly Ser Gly Gly Asp Glu Thr Val Pro Thr Asp Ser Gly

515 520 525

Gln Ala Pro Ala Leu Ala Gly Val Lys Ala Ser Phe Arg His Gly Ser

530 535 540

Lys Gly Lys Gly Gln Ala Asn Thr Lys Arg Gly Tyr Glu His Gln Glu

545 550 555 560

Ser Tyr Asn Asp Ala Arg Ala Gln Trp Ala Ala Leu Tyr Gly Val Ile

565 570 575

Lys Ile Thr Gln Leu Ala Asp Trp His Pro Asn Asp Lys Gly Gly Thr

580 585 590

Met Met His Pro Arg Arg Leu Leu Ala Val Leu Gly Val Val Val Leu

595 600 605

Thr Ser Met Thr Ser Cys Thr Ser Phe Ser Ser Ser Arg Ser Ser Ser

610 615 620

Met Asp Lys Thr Gly Trp Ile Thr His Cys Phe Gly Arg Phe Leu Ile

625 630635 640

Asp Leu Pro Pro Asp Ala Val Ile Asn Ala Gly Tyr Tyr Leu Trp Gly

645 650 655

Asp Arg Ile Glu Tyr Leu Asp Asp Lys Pro Thr Glu Leu Ala Ala Arg

660 665 670

Val Asp Arg Leu Glu Gln Glu Trp Arg Thr Gln Arg His Lys Ser Lys

675 680 685

Gly Asn Met Phe Leu Arg Lys Ile Asp Phe Gly Asn Glu Ser Val Gly

690 695 700

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

705 710 715 720

Thr Tyr Val Thr Ser Lys Pro Thr Trp His Val Tyr Arg Trp Lys Gly

725 730 735

Lys Val Ser Val Asp Arg Glu Gln His Ala Val Glu Ile Ser Arg Ala

740 745 750

Leu Ala Arg Asn Leu Arg Ser Arg Ala Pro Lys Glu Ile Pro Ser Glu

755 760 765

Pro Gly Phe Cys Ile Asp His Ala Tyr Ile Ala Gly Asp Ser Phe Gln

770 775 780

Val Glu Arg Phe Gly Val Gly Val Thr Phe Pro Glu His Pro Gly Ala

785 790795 800

Arg Phe Glu Phe Arg Ser Ser Thr Gly Ala Glu Leu Asn Ser Leu Leu

805 810 815

Glu Arg Val Asp Gly Phe Val Gln Asn Met Leu Ser Thr Phe Ala Gly

820 825 830

Met Glu Thr Leu Arg Lys Gly Lys His Pro Val Gly Ser Leu Pro Gly

835 840 845

Glu Glu Tyr Leu Val Ala Gly Ser Asp Lys Gly Gln Arg Gly Tyr Thr

850 855 860

Phe Met Trp Glu Val Gln Gly Lys Glu Glu Ser Leu Thr Glu Pro Asn

865 870 875 880

Leu Thr Ala Gly Leu Ala Val Leu Glu Arg Ser Asn Glu Asn Gly Lys

885 890 895

Pro Pro Pro Pro Ala Phe Lys Ser Asp Lys Glu Ala Leu Glu Leu Trp

900 905 910

Asp Thr Ile Val Asp Ser Ile Arg Val Arg Pro Thr Ser Ser Ser Pro

915 920 925

Arg Gly Gly Asn Ala Gly Pro Ser Pro Ala Pro Lys Pro Ala Thr Pro

930 935 940

Gly Gly Gln Thr Leu Gly Asp His Tyr Val Tyr Glu Glu Phe Leu Ser

945 950 955960

Ser Leu Lys Pro Lys Asp Ser Trp Leu Asp Asp Leu

965 970

<210>10

<211>198

<212>DNA

<213>Artificial sequence

<400>10

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60

atggccatgg atgacctgtt tgcctccatc ggtgcgttgt ggacttgggc ctggaggggt 120

ccaaaggccc gtcaggagct cctgaaggct gagcaagtag aagtcgatga cctcgagcac 180

caccaccacc accactga 198

<210>11

<211>65

<212>PRT

<213>Artificial sequence

<400>11

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala

1 5 10 15

Ala Gln Pro Ala Met Ala Met Asp Asp Leu Phe Ala Ser Ile Gly Ala

20 25 30

Leu Trp Thr Trp Ala Trp Arg Gly Pro Lys Ala Arg Gln Glu Leu Leu

35 40 45

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

50 55 60

His

65

<210>12

<211>198

<212>DNA

<213>Artificial sequence

<400>12

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60

atggccatgg atgatctgtt tgccagcatt ggcgcactgt ggacctgggc atggcgtggt 120

ccggaagcac gtcaggaact gctgaaagcc gaacaggtgg aagtggatga cctcgagcac 180

caccaccacc accactga 198

<210>13

<211>198

<212>DNA

<213>Artificial sequence

<400>13

atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg 60

atggccatgg atgatctgtt tgccagcatt ggtgcactgg caacctgggc atggcgtggt 120

ccgaaagccc gtcaggaact gctgaaagcc gaacaggtgg aagtggatga tctcgagcac 180

caccaccacc accactga 198

Claims

1. A polypeptide which is (a1), (a3), (a4), (a5), (a6), (a7) or (a 8):

(a1) polypeptide shown in sequence 3 of the sequence table;

(a3) polypeptide shown in sequence 4 of the sequence table;

(a6) 1 st to 57 th amino acid residues of a sequence 11 in a sequence table;

(a8) polypeptide shown in sequence 11 of the sequence table.

2. A gene encoding the polypeptide of claim 1.

3. The gene of claim 2, wherein: the genes are (b1) or (b2) or (b3) or (b4) as follows:

(b4) the coding region is shown as a DNA molecule in a sequence 10 of a sequence table.

4. A recombinant expression vector, expression cassette, transgenic cell line or recombinant bacterium comprising the gene of claim 2 or 3.

5. The use of the polypeptide of claim 1, which is (c2) or (c4) or (c 6):

6. The use of the polypeptide of claim 1, which is as follows: a product is prepared for competitive binding of the immunity protein to the toxin protein in the bacterial type vi secretion system.

7. The use of the polypeptide of claim 1, which is as follows: preparing a product for relieving the inhibition of bacterial death induced by toxin proteins by immune proteins in a bacterial type VI secretion system.

8. The use of the polypeptide of claim 1, which is as follows: preparing a product for promoting the death of bacteria having a bacterial type VI secretion system.