CN114262366B - B cell epitope polypeptide HP11 of helicobacter pylori HspA and application thereof - Google Patents

Abstract

The invention discloses a B cell epitope polypeptide HP11 of helicobacter pylori HspA and application thereof. The invention provides an HspA epitope peptide, which is 1) or 2): 1) A polypeptide shown in the 2-12 positions of the sequence 1; 2) To add a tag sequence at the end of the sequence of the polypeptide shown in 1). The invention identifies a new B cell epitope HP11 from HspA, detects the antibody expression profile of the epitope peptides in H.pyri natural infected people, and provides a basis for further researching the effect of HspA in H.pyri infection immunodiagnosis and immunoprophylaxis. Antibodies prepared from the epitope peptide can be used to detect HspA or H.pyri infection.

Description

B cell epitope polypeptide HP11 of helicobacter pylori HspA and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a B cell epitope polypeptide HP11 of helicobacter pylori HspA and application thereof.

Background

In 1982, two australians Robin Warren and barre Marshall isolated helicobacter pylori from human gastric tissue (Helicobacter pylori, h. Pyrri) and elucidated the relationship of this bacteria to gastrointestinal disease. In 2005, the nobel physiology or medical prize awarded to the two scientists in australia to bring out their contribution to this.

Pyrri is a spiral, microaerophilic, gram-negative bacterium that colonizes the human stomach. The discovery of pyri has led to subversive changes in the pathogenesis and prevention of chronic gastritis, peptic ulcer, and other diseases. At present, H.pyri infection is widely accepted by the International medical community as the most important cause of chronic gastritis and peptic ulcer, and there is enough evidence that the bacterial infection has carcinogenicity to human beings and is highly related to gastric adenocarcinoma and occurrence of mucosa-associated lymphoid tissue (MALT) lymphoma. In 1994, WHO international cancer research Institute (IARC) and NIH in the united states sequentially announced, respectively, by resolution: pyrori is the causative agent of gastritis, gastric and duodenal peptic ulcers, and is also a first type of carcinogen. To date, h.pyri is the only pathogenic bacteria recognized to be associated with the pathogenesis of cancer.

The h.pyri infection is globally distributed with an infection rate of over 50% of the world's natural population. In developing countries, 50% -80% or more of adults are infected with this bacterium; in developed countries, the infection rate of H.pyri is also 25% to 50%. In China, according to epidemiological investigation, the H.pyri infection rate is 40% -90%, and the average is 59%; the lowest region is Guangdong, 42%; the highest area is the Tibet, 90%. The infection rate of H.pyri in China is 42-64%, and the average rate is 55%; the lowest region is Guangdong, 42%; the highest region is Shaanxi, 64%. The infection rate of children H.pyri in China is 25-59%, 40% on average, and the infection rate increases at a rate of 0.5% -1% per year on average.

Once obtained, h.pyrri continues to colonize and grow in the human stomach for years, decades, or even lifetime if untreated. Asymptomatic gastritis appears in 100% of infected persons, about 30% of infected persons develop chronic gastritis, 10-20% of infected persons develop gastric and duodenal peptic ulcers, and about 1% -2% of infected persons develop gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphomas. Clinical tests show that the detection rate of the chronic gastritis H.pyri is 54-100%, the detection rate of the chronic active gastritis H.pyri is more than 90%, the detection rate of the gastric ulcer H.pyri is more than 80%, and the detection rate of the duodenal ulcer H.pyri is more than 90%. H.pyri infected subjects have 6-8 times the incidence of gastric cancer than non-infected subjects and h.pyri infected subjects have 20 times the incidence of young gastric cancer than non-infected subjects. H.pyri has also been shown to be involved in many parenteral diseases, such as those associated with oral, skin, blood, cardiovascular and respiratory systems and even pregnancy, and pediatric diseases, in addition to the diseases mentioned above.

Based on the recognition of H.pyri infection-induced diseases, the common and multiple diseases of the digestive tract are known to be treated by eradicating H.pyri. The currently employed treatment is based on a triple or quadruple combination of antibiotics, proton pump inhibitors and bismuth compounds, with a course of treatment lasting 10-14 days. However, over the last decade, with the rapid increase in resistance to clarithromycin in many countries, the eradication rate of H.pyri has declined worldwide. In addition to the increasing reports of drug-resistant strains, the high treatment cost, poor drug compliance and high recurrence rate of infection are also the main causes of failure in large-scale control of h.pyri infection. Thus, the study of immune response and immune protection mechanisms against h.pyri is of positive interest for overcoming the problems with current therapeutic approaches, more effective detection and assessment, and even prevention and treatment of h.pyri infection.

H.pyri heat shock protein a (heat shock protein A, hspA) is a bacterial heat shock chaperone, one of the key virulence factors and protective antigens for h.pyri infection. HspA consists of 118 amino acids and is divided into two domains: the A domain (amino acids 1-90), which has sequence similarity to GroES sequences, and the B domain (amino acids 91-118), which is unique to H.pyri and Helicobacter acinonuchis, contains 8 histidines and 4 cysteines. HspA is involved in intracellular nickel sequestration and transport, maintaining Ni2 of H.pyri ⁺ And (3) steady state. Metallic nickel can affect H.pyri urease and [ NiFe]Activity and function of hydrogenase, two enzymes are important virulence factors for h.pyri survival and colonization in the stomach. HspA plays an important role in the diagnosis, treatment and prevention of h. About 40% of h.pyri infected persons can detect the HspA antibody. Bi (Bi) ³⁺ Irreversibly binds to HspA and interferes with its biological function, which is the primary mechanism of therapeutic effect of bismuth compounds. HspA has significant immunoprotection against H.pyri in a mouse modelActing as a medicine.

It is well known that the body resists invasion and pathogenesis of foreign pathogens by recognizing epitopes of pathogenic bacteria and generating an adaptive immune response. Therefore, the identification of the epitope has important significance for immunodiagnosis of infectious diseases, study of immune protection mechanism and research of vaccine development.

Disclosure of Invention

The invention aims to provide an HspA epitope peptide.

The HspA epitope peptide provided by the invention is 1) or 2):

1) A polypeptide shown in the 2-12 positions of the sequence 1;

2) To add a tag sequence at the end of the sequence of the polypeptide shown in 1).

It is another object of the present invention to provide an HspA antigen.

The antigen provided by the invention is formed by coupling the HspA epitope peptide with carrier protein.

Among the above antigens, the carrier protein is keyhole limpet hemocyanin or bovine serum albumin.

The application of the HspA epitope peptide or the HspA antigen as an immunogen to prepare an anti-HspA antibody is also within the protection scope of the invention.

Antibodies against HspA prepared from the above-described HspA epitope peptide or the above-described HspA antigen as an immunogen are also within the scope of the present invention.

The use of the antibodies described above is also within the scope of the present invention as follows:

1) Detection or assisted detection of HspA;

2) Detection or assisted detection of h.pyri;

3) Preparing a product for detecting or assisting in detecting HspA;

4) Use in the manufacture of a product for the detection or co-detection of a disease associated with h.pyri infection.

The use of the above-mentioned HspA epitope peptide or the above-mentioned HspA antigen or the above-mentioned antibody or a substance containing the above-mentioned HspA epitope peptide as an active ingredient in any one of the following (a) to (d) is also within the scope of the present invention:

(a) Preparing a product for diagnosing or assisting in diagnosing diseases related to abnormal expression of HspA;

(b) Preparing a product for preventing diseases related to abnormal expression of HspA;

(c) Preparing a product for diagnosing or aiding in diagnosing a disease associated with H.pyrri infection;

(d) A product for preventing diseases related to the induction of h.pyri infection is prepared.

The product for preventing the diseases related to abnormal expression of HspA is a vaccine, and can be a multi-linked multimeric epitope vaccine preparation.

It is also an object of the invention to provide a product.

The active ingredients of the product provided by the invention are the above HspA epitope peptide or the above HspA antigen or the above antibody;

the product has at least one of the following functions:

(a) For diagnosing or aiding in diagnosing diseases associated with abnormal expression of HspA;

(b) For preventing diseases associated with abnormal expression of HspA;

(c) For diagnosis or assisted diagnosis of h.pyri infection-induced related diseases;

(d) For preventing H.pyri infection-induced related diseases.

Nucleic acid molecules encoding the above-described HspA epitope peptides are also within the scope of the present invention.

Expression cassettes, recombinant vectors or recombinant cell lines containing the above-described nucleic acid molecules are also within the scope of the present invention.

The invention identifies a new B cell epitope HP11 from HspA, detects the antibody expression profile of the epitope peptides in H.pyri natural infected people, and provides a basis for further researching the effect of HspA in H.pyri infection immunodiagnosis and immunoprophylaxis. Antibodies prepared from the epitope peptide can be used to detect HspA or H.pyri infection.

Drawings

FIG. 1 shows the selection of peptide fragments of the immunodominant reaction of HspA antigen. (A) Truncated fragment of HspA from h. (B) OD values of five peptide fragments detected by ELISA with 20 mouse anti-rHspA sera.

FIG. 2 shows the epitope fine localization of HP1 peptide fragment in HspA. (A) overlapping synthetic peptides covering HP 1. Each peptide stretch comprises 11 amino acids, 9 of which overlap with adjacent peptide stretches. (B) ELISA detection results of 11 polypeptides and mouse anti-rHspA serum.

FIG. 3 shows the immunogenicity and immunoreactivity of the HP11 and HP19 epitopes. (A) ELISA detection results of antigen epitope-KLH mouse antiserum and synthetic epitope peptide. The synthesized epitope peptides HP11 and HP19 are used as coating antigens, the antigen epitope-KLH mouse antiserum is used as a primary antibody, and PBS immunized mouse serum is used as a negative control. (B) immunoblotting analysis of the HP 11-KLH-immunized mouse antiserum. (C) immunoblotting analysis of HP 19-KLH-immunized mouse antiserum. (D) immune mouse antiserum immunoblotting analysis of rHspA.

FIG. 4 shows epitope-specific lymphocyte proliferation responses. The epitope stimulates the spleen lymphocytes of the corresponding HP11-KLH (A) and HP19-KLH (B) immunized mice to undergo proliferation. The antigen epitope stimulated PBS immunized mice spleen lymphocytes as a negative control, conA stimulation as a positive control. Splenic lymphocyte stimulation index (stimulation index, SI) is the OD of the stimulated wells divided by the OD of the control wells. * P <0.05, < P <0.01vs. negative control.

FIG. 5 is a serum antibody profile of epitopes HP11 (A) and HP19 (B) in naturally infected individuals. Serum antibody OD values against epitopes HP11 and HP19 were detected by ELISA with 42 HspA seropositive samples from h.pyri infected patients. 17 H.pyri antibody negative serum was used as negative control. Positive determination criteria: OD > negative control serum mean OD 2.1.

Detailed Description

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Bacterial strains and culture conditions in the following examples:

in the following examplesThe supernatant of the ultrasonic fragmentation of h.pyrriss 2000 was prepared as follows: h.pyri Strain Sydney Strain 2000 (SS 2000) (Thompson LJ, danon SJ, wilson JE, et al, chronic Helicobacter pylori Infection with Sydney Strain 1AND a Newly Identified Mouse-Adapted stress (Sydney stress 2000) in C57BL/6AND BALB/C Mice.INFECTION AND IMMUNITY,2004,72 (8): 4668-4679) AND NCTC11637 (U.S. ATCC, ATCC 43504) on H.pyri-selective agar plates (Campylobacter Agar Base) containing 7% fetal bovine serum at 37℃5% O ₂ 、10％CO ₂ And 85% N ₂ Is cultured under microaerophilic conditions. After 3 days of incubation, colonies were scraped and washed twice with pre-chilled Phosphate Buffer (PBS) at pH7.0 at 20mM, and then centrifuged at 10,000Xg for 10 minutes at 4℃to obtain bacterial pellet. H.pyri SS2000 pellet was resuspended in PBS, sonicated by ice bath (300 w,10s,20 min), centrifuged for 20min, and the H.pyri SS2000 supernatant was collected as the supernatant containing HspA for Western Blotting (WB). Bacterial precipitation of H.pyri NCTC11637, and extraction of NCTC11637 genomic DNA using a bacterial genomic DNA extraction kit (Beijing Tian Gen Biotechnology Co., ltd.).

The construction, expression and purification methods of H.pyri recombinant HspA (rHspA) and GST fusion peptides in the following examples are as follows:

the gene sequence of HspA (SEQ ID NO: 2) was amplified directly from the genome of H.pylori strain NCTC11637 by PCR and cloned into pGEX-6P-1 (+) (GE Healthcare) expression vector, placed between BamHI and NotI or XhoI cleavage sites, to obtain recombinant plasmids. The recombinant plasmid was transformed into E.coli BL21 (DE 3) pLysS cells (Cwbio) and 1mM IPTG was used to induce expression of the recombinant fusion protein GST-HspA. Cells were collected by centrifugation and bacterial pellet resuspended in PBS, the resuspended cells were sonicated, and the supernatant collected by centrifugation, i.e., containing the recombinantly expressed GST-HspA fusion protein.

Recombinant GST-HspA fusion protein was purified by Price Glutathione Superflow Agarose (Thermo). Then, GST tag was cleaved with precision protein enzyme (Beyotime) and removed using GST tag purification resin (Beyogold), and purified according to the instructions (GE Healthcare, USA)，Recombinant protein rHspA is obtained.

The purified recombinant protein rHspA was analyzed by SDS-PAGE, and the concentration of the purified protein was determined by BCA method.

GST-HP11 and GST-HP19 are constructed, expressed and purified similarly to GST-HspA fusion proteins, except that the coding gene sequence of HspA is replaced with the coding gene sequence corresponding to the HP11 or HP19 polypeptide, and the GST tag is not removed.

In the following examples, the synthesis of HP1-HP5 polypeptides, the synthesis of overlapping peptides covering the entire length of HP1, and the conjugation of polypeptide HP11 to HP19 with Keyhole Limpet Hemocyanin (KLH) (preparation of HP11-KLH conjugated peptide or HP19-KLH conjugated peptide) were all accomplished by GenScript (Nanj, china). Standard solid phase FMOC methods were used for peptide synthesis, with purity >95% assessed by high pressure liquid chromatography and identified by laser desorption mass spectrometry. Peptides were dissolved in DMSO or ultra-pure water at a concentration of 10mg/ml and stored in aliquots at-20 ℃.

The ELISA detection method in the following examples was as follows:

when coating with polypeptide as antigen, 96-well ELISA plates (Costar) were first pretreated with 150. Mu.l of 2.5% glutaraldehyde for 1 hour at 37℃and then washed four times with water. The epitope peptide was diluted as coating antigen in 0.1mM carbonate buffer (pH 9.6) and ELISA plates (30. Mu.g/ml, 100. Mu.l/well) were coated and incubated overnight at 37 ℃. When HspA was used as antigen for coating, the antigen was diluted in 0.1mM carbonate buffer (pH 9.6) and ELISA plates (2. Mu.g/ml, 100. Mu.l/well) were coated and incubated overnight at 4 ℃. Plates were washed 3 times with PBST.

ELISA plates were added with blocking buffer (PBST containing 5% skimmed milk for mouse serum, protein free blocking solution (Thermo) for human serum), 200 μl/well, and blocked for 1 hour at 37deg.C. Blocking solution was removed, 100. Mu.l of rHspA immune serum (1:500) or KLH binding peptide immune serum (1:100) or H.pyri positive serum (1:100) was added to each well and incubated for 1 hour at 37 ℃. PBS immunized mice or h.pyri infection negative healthy human serum was used as negative control.

After washing wells 3 times with PBST, HRP conjugated rabbit anti-mouse IgG (1:5,000 dilution, abcam) or goat anti-human IgG (1:20,000 dilution, abcam) secondary antibodies were added to the wells (100. Mu.l/well) The ELISA plates were washed as above after incubation at 37℃for 1 hour. The enzyme substrate solution 3, 5-tetramethylbenzene-biphenyl (TMB) was added at 100. Mu.l/well and the reaction was carried out at room temperature for 15 minutes. With 100 μl of 1MH ₂ SO ₄ The reaction was terminated and absorbance at 450nm (A450) was measured with ELISA plate reader. All assays were performed with multiple wells and the assay results were averaged with multiple wells.

The rHspA antiserum in the examples below was prepared by immunizing 6-8 week old female SPF BALB/c mice (20 animals per group at the center of laboratory animals of the military medical institute) subcutaneously with 50. Mu.g of rHspA in emulsion with complete Freund's adjuvant (Sigma). Immunization was boosted with 50 μg of rHspA in emulsion with incomplete freund's adjuvant (Sigma) on days 10 and 20 post-immunization. Mice were sacrificed on day 30 and serum samples were collected as anti-rHspA serum.

Statistical analysis in the examples below, all data are expressed as mean ± standard deviation (s.d.). The mean values were compared using a two-tailed t-test and analyzed using GraphPad prism8.0.2 (GraphPad Software), P <0.05 was considered statistically significant.

Example 1 identification and preparation of HspA epitope peptide and detection of immunogenicity and immunoreactivity

1. Screening of HspA antigen immunodominant reaction peptide fragment

Shortening HspA (amino acid sequence is sequence 1 in a sequence table) into five sections, wherein the first 4 peptide sections are positioned in an A domain of HspA, 8 amino acids are overlapped on each section, and the sections are named HP1-HP4 from the N end to the C end in sequence; the 5 th peptide fragment is located in the B domain, overlapping HP4 by 2 amino acids (FIG. 1A).

The amino acid sequence of the HP1 peptide fragment is the 2 nd-31 rd position of the sequence 1;

the amino acid sequence of the HP2 peptide fragment is 34-53 of the sequence 1;

the amino acid sequence of the HP3 peptide fragment is 46-75 of the sequence 1;

the amino acid sequence of the HP4 peptide fragment is 68-91 of the sequence 1;

the amino acid sequence of the HP5 peptide fragment is 90-118 of the sequence 1;

the five polypeptides were synthesized as antigens and tested by ELISA with 20 mouse anti-rHspA sera (HspA antisera).

The results are shown in FIG. 1B, which shows that HP1 can react with all antisera, the immune response is strongest, and HP1 is an HspA antigen immunodominant reaction segment.

2. Fine localization of HP1 epitopes

In order to fine-locate the epitope of the HP1 peptide fragment in HspA, a set of 11 amino acid overlapping polypeptides was synthesized, covering amino acids 2-31 of HspA, giving a total of 11 polypeptides (FIG. 2A).

The amino acid sequence of the HP11 polypeptide is the 2 nd-12 th positions of the sequence 1;

the amino acid sequence of the HP12 polypeptide is the 4 th-14 th positions of the sequence 1;

the amino acid sequence of the HP13 polypeptide is the 6 th-16 th positions of the sequence 1;

the amino acid sequence of the HP14 polypeptide is 8-18 of the sequence 1;

the amino acid sequence of the HP15 polypeptide is the 10 th to 20 th positions of the sequence 1;

the amino acid sequence of the HP16 polypeptide is the 12 th-22 th positions of the sequence 1;

the amino acid sequence of the HP17 polypeptide is 14 th-24 th of the sequence 1;

the amino acid sequence of the HP18 polypeptide is from 16 th to 26 th of the sequence 1;

the amino acid sequence of the HP19 polypeptide is 18-28 of the sequence 1;

the amino acid sequence of the HP20 polypeptide is 20-30 of the sequence 1;

the amino acid sequence of the HP21 polypeptide is the 21 st-31 st position of the sequence 1;

the above polypeptides were synthesized, ELISA plate coating was performed using the synthesized polypeptides as coating antigens, and ELISA method was used to detect the reactivity of 11 polypeptides with 20 mouse anti-rHspA serum (HspA antiserum).

As a result, as shown in FIG. 2B, it can be seen that HP11, HP18, HP19, and HP20 are capable of generating a strong immune response. Of these, the three peptides HP18, HP19 and HP20 are similar in sequence, and HP19 (ENKTSSGIIIP) is the most immunoreactive, presumably HP19 is an epitope of HspA. Furthermore, the immune response of HP11 (KFQPLGERVLV) is comparable to that of HP20, possibly another epitope of HspA.

3. Immunogenicity and immunoreactivity assays for HP11 epitope peptides and HP19 epitope peptides

In the preliminary determination of two B-cell epitopes in HspA, it was first assessed whether these epitopes were able to induce an immune response in mice, as follows:

1. KLH-conjugated peptides of HP11 (HP 11-KLH) and HP19 (HP 19-KLH)

To enhance the immunogenicity of the polypeptides, a cysteine was attached to the C-terminus of HP11 and HP19, which were then coupled to KLH using the MBS method to form KLH-coupled peptides.

The HP11-KLH conjugated peptide is a polypeptide obtained by connecting a cysteine to the C-terminal of HP11, and then the HP11 with the cysteine connected to the C-terminal is conjugated to the KLH to form the HP11-KLH conjugated peptide (the feeding mass ratio of the HP11 with the cysteine connected to the C-terminal and the KLH is 1:1).

The HP19-KLH conjugated peptide is a polypeptide obtained by connecting a cysteine to the C-terminal of HP19, and then the HP19 with the cysteine connected to the C-terminal is conjugated to KLH to form the HP19-KLH conjugated peptide (the feeding mass ratio of the HP11 with the cysteine connected to the C-terminal and the KLH is 1:1).

2. Mouse immunity and sample collection

Immunogenicity of rHspA and HP 11-KLH-conjugated peptide or HP 19-KLH-conjugated peptide was assessed by immunization of 6-8 week old female SPF BALB/c mice (10-20 animals per group at the laboratory animal center of the military medical institute).

Primary immunization was performed subcutaneously using 50. Mu.g rHspA or 50. Mu.g HP11-KLH conjugated peptide or 50. Mu.g HP19-KLH conjugated peptide in emulsion mixed with complete Freund's adjuvant (Sigma), respectively.

On days 10 and 20 after priming, booster immunizations were performed using emulsions prepared by mixing the same kind and dose of antigen as the priming with incomplete Freund's adjuvant (Sigma), respectively.

Control mice were immunized with PBS using the same immunization program.

Mice were sacrificed on day 30, serum samples were collected to obtain rHspA immune serum, HP11-KLH conjugate peptide immune serum, HP19-KLH conjugate peptide immune serum, and analyzed by ELISA and western immunoblotting; collecting spleen tissues to obtain rHspA immune mouse spleen cells, HP11-KLH coupled peptide immune mouse spleen cells and HP19-KLH coupled peptide immune mouse spleen cells, and carrying out lymphocyte proliferation test.

All animals were purchased from velocin rituximab (beijing, china) and raised under SPF conditions.

3. ELISA for detecting immunogenicity of antigen epitope

The HP11 polypeptide and the HP19 polypeptide were used as detection antigens for coating, the HP 11-KLH-conjugated peptide immune serum (1:100, also called HP11-KLH antiserum) and the HP 19-KLH-conjugated peptide immune serum (1:100, also called HP11-KLH antiserum) of the mice collected in the above 2 were used as antibodies, and all sera obtained from the KLH-conjugated peptide immunized mice were detected by ELISA. PBS immunized mouse serum was used as a negative control.

As shown in FIG. 3A, the mice were induced to develop a strong immune response by injecting HP 11-KLH-conjugated peptide and HP 19-KLH-conjugated peptide, and the titers of the 2 epitope peptide-specific antibodies (antisera) were all over 1:1000. both epitopes were able to react ELISA positive with the serum of mice immunized with KLH-conjugated peptide by themselves, and neither epitope was able to react ELISA positive with the serum of mice immunized with KLH-conjugated peptide or PBS by the other epitope (fig. 3A).

4. Immunoblotting analysis method for detecting immunoreactivity of epitope

Western blot analysis of serum immunized with KLH conjugated peptide with h.pyri SS2000 supernatant, rHspA and GST fusion protein was performed as follows:

preparing an H.pyrri SS2000 sonicated supernatant according to the previous method;

the amino acid sequence of the rHspA protein is the protein with the sequence 1.

The amino acid sequence of GST fusion expression protein GST-HP11 is sequence 3, wherein the 1 st to 229 th positions of the sequence 3 are GST labels, the 230 st to 231 th positions are Linker, and the 232 nd to 242 nd positions are HP11.

The amino acid sequence of GST fusion expression protein GST-HP19 is sequence 4, wherein the 1 st to 229 th positions of the sequence 4 are GST labels, the 230 st to 231 th positions are Linker, and the 232 nd to 242 nd positions are HP19.

rHspA protein is used as positive control, GST protein is used as negative control.

H.pyrri SS2000 sonicate supernatant (SS 2000), rHspA, GST-HP11 fusion protein, GST-HP19 fusion protein and GST were separated by 15% SDS-PAGE under denaturing conditions, transferred to a transfer membrane (Merck Millpore) and blocked with blocking buffer (5% skim milk in PBST, pH 7.4)) for 1 hour at 37℃to prevent non-specific protein binding. The membranes were incubated with mouse HP11-KLH conjugated peptide immune serum (1:500 dilution) or HP19-KLH conjugated peptide immune serum (1:500 dilution) collected with 2 above for 1 hour at 37℃and washed 3 times with PBST at room temperature. HRP conjugated rabbit anti-mouse IgG (1:5,000 dilution, abcam) was reacted at 37 ℃ for 1 hour and detected using HRP western blot analysis kit (Easybio, china).

As a result, as shown in FIGS. 3B and 3C, the HP11-KLH immune serum (HP 11-KLH antiserum) and the HP19-KLH immune serum (HP 19-KLH antiserum) can generate immunoblotting reaction against H.pyri SS2000 native HspA (SS 2000), rHspA expressed in E.coli and its own GST fusion protein (GST-HP 11 or GST-HP 19), but cannot react with GST (FIGS. 3B and 3C).

The antisera prepared from mice immunized with rHspA were then immunoblotted with native HspA from SS2000, rHspA protein, and the two GST fusion proteins GST-HP11 and GST-HP 19. The results demonstrate that both fusion proteins GST-HP11 and GST-HP19, along with SS2000 native HspA and rHspA proteins, can immunoblotted with rHspA antisera, whereas GST alone does not react with rHspA antisera (FIG. 3D).

5. Lymphocyte proliferation response assay

To further understand the epitope-induced immune response, the epitope-stimulated specific lymphocyte proliferation activity from the epitope-conjugated KLH peptide immunized mouse spleen lymphocytes was examined. Spleen cells of HP11-KLH or HP19-KLH immunized mice were stimulated with the corresponding epitope, while PBS immunized mice were stimulated with the same epitope as a negative control, and Canavalia A (ConA, solarbio, cat# C8110) was stimulated as a positive control. The method comprises the following steps:

using injectionThe mouse spleen tissue was immunized with the HP11-KLH conjugated peptide prepared in the above 2, and the mouse spleen cell spleen tissue was immunized with the HP19-KLH conjugated peptide, and the nylon mesh was finely crushed to prepare a single cell suspension. Cells were seeded in 96-well cell culture plates in RPMI-1640 medium (Gibco, U.S.A.) containing 10% FBS, 4X 10 cells per well ⁵ Individual cells. Under stimulation conditions without any stimulus (negative control) and with 10. Mu.g/mL of epitope peptide HP11 polypeptide or HP19 polypeptide or 0.625. Mu.g/mLConA (corresponding concentration of epitope peptide or ConA added to the cells), 5% CO at 37 ℃C ₂ Culturing for 72 hours under the condition. Mice immunized with PBS served as a negative control group (negative control). During the last four hours of incubation, 20 μ l Cell Counting Kit-8 (CCK 8, dojindo, japan) solution was added to each well and a450 was measured using SpectraMaxi3x (Molecular Devices, usa). The splenic lymphocyte Stimulation Index (SI) is the OD of the stimulated wells divided by the OD of the control wells.

As a result, as shown in FIG. 4, conA stimulated the spleen cells of mice to develop nonspecific strong proliferation, and the antigen epitope peptide stimulated the corresponding antigen epitope peptide-KLH conjugate peptide immunized mice had significantly higher proliferation activity than the negative control group (P <0.01, FIG. 4).

4. Antibody detection of epitope of natural infectious agent

Antibody expression profiles of two B cell epitopes identified in HspA were tested with serum from h.pyri natural infected persons.

The study was approved by the fourth medical center ethics committee of the release army general hospital and informed consent was obtained from the patients. Hospitalized patient serum was collected and tested for the presence of anti-h.pyri antibodies using the h.pyri IgG ELISA kit (IBL). The experimental procedure was performed according to the kit instructions. Serum was considered to be h.pyri positive when the critical index (COI) >1.2, and h.pyri negative when the critical index (COI) < 0.8.

85 H.pyri positive sera (designated as P1-P85) were selected and tested for HspA antibodies and epitope antibodies by ELISA, and the coating antigens were rHspA, epitope peptides HP11 and HP19, respectively, and 17 H.pyri antibody negative sera were used as negative controls. The positive judgment limits of the HspA antibody and the epitope antibody are as follows: OD > negative control serum mean OD 2.1.

The results were as follows: ELISA detection of 42 HspA antibodies positive in 85 H.pyri infected patients showed a positive rate of 49.4%. Negative serum (N1-N17) was detected by the H.pyri IgG ELISA kit as a negative control. Of 42 HspA seropositive patients, 9 detected positive antibodies against the HP11 epitope, and 14 detected positive antibodies against the HP19 epitope, with positive rates of 21.4% and 33.3%, respectively. The lower panel shows the antibody response profile for two epitopes in HspA seropositive patients (figure 5).

SEQUENCE LISTING

<110> military medical institute of the military academy of China's civil liberation army

<120> B cell epitope polypeptide HP11 of helicobacter pylori HspA and application thereof

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 118

<212> PRT

<213> Artificial sequence

<400> 1

Met Lys Phe Gln Pro Leu Gly Glu Arg Val Leu Val Glu Arg Leu Glu

1 5 10 15

Glu Glu Asn Lys Thr Ser Ser Gly Ile Ile Ile Pro Asp Asn Ala Lys

20 25 30

Glu Lys Pro Leu Met Gly Val Val Lys Ala Val Ser His Lys Ile Ser

35 40 45

Glu Gly Cys Lys Cys Val Lys Glu Gly Asp Val Ile Ala Phe Gly Lys

50 55 60

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

65 70 75 80

Glu Leu Glu Asp Ile Leu Gly Ile Val Gly Ser Gly Ser Cys Cys His

85 90 95

Thr Gly Asn His Asp His Lys His Ala Lys Glu His Glu Ala Cys Cys

100 105 110

His Asp His Lys Lys His

115

<210> 2

<211> 357

<212> DNA

<213> Artificial sequence

<400> 2

atgaagtttc aaccattagg agaaagggtc ttagtagaaa gacttgaaga agagaacaaa 60

accagttcag gcattatcat ccctgataac gctaaagaaa agcctttgat gggcgtagtc 120

aaagcggtta gccataaaat cagtgagggt tgcaaatgcg tcaaagaagg cgatgtgatc 180

gcttttggca aatataaagg tgcagaaatc gttttagatg gcaccgaata catggtgcta 240

gaactagaag acattctggg cattgtgggt tcaggctctt gttgtcatac aggtaatcat 300

gatcataagc atgctaaaga gcatgaagct tgctgtcatg atcacaaaaa acactaa 357

<210> 3

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<212> PRT

<213> Artificial sequence

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Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu

210 215 220

Phe Gln Gly Pro Leu Gly Ser Lys Phe Gln Pro Leu Gly Glu Arg Val

225 230 235 240

Leu Val

<210> 4

<211> 242

<212> PRT

<213> Artificial sequence

<400> 4

Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro

1 5 10 15

Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu

20 25 30

Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu

35 40 45

Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys

50 55 60

Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn

65 70 75 80

Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu

85 90 95

Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser

100 105 110

Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu

115 120 125

Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn

130 135 140

Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp

145 150 155 160

Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu

165 170 175

Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr

180 185 190

Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala

195 200 205

Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu

210 215 220

Phe Gln Gly Pro Leu Gly Ser Glu Asn Lys Thr Ser Ser Gly Ile Ile

225 230 235 240

Ile Pro

Claims (7)

1. HspA epitope peptide is polypeptide shown in the 2 nd-12 th positions of a sequence 1.
2. An HspA antigen, which is formed by coupling the HspA epitope peptide of claim 1 with a carrier protein; the carrier protein is keyhole limpet hemocyanin.
3. 3. Use of the HspA epitope peptide of claim 1 or the HspA antigen of claim 2 as an immunogen for the preparation of an anti-HspA antibody.
4. 4. Use of a substance comprising the HspA epitope peptide of claim 1 as an active ingredient in any one of the following (a) - (d):

   (a) Preparing a product for diagnosing or assisting in diagnosing diseases related to abnormal expression of HspA;

   (b) Preparing a product for preventing diseases related to abnormal expression of HspA;

   (c) Preparation for diagnosis or auxiliary diagnosisH.pyloriProducts that cause infection-related diseases;

   (d) Preparation for preventionH.pyloriProducts that cause the associated disease by infection.
5. 5. Use of the HspA epitope peptide of claim 1 or the HspA antigen of claim 2 for the preparation of a product having at least one of the following functions;

   (a) For diagnosing or aiding in diagnosing diseases associated with abnormal expression of HspA;

   (b) For preventing diseases associated with abnormal expression of HspA;

   (c) For diagnosis or auxiliary diagnosisH.pyloriInfection causes related diseases;

   (d) For preventingH.pyloriInfection causes related diseases.
6. 6. A nucleic acid molecule encoding the HspA epitope peptide of claim 1.
7. 7. An expression cassette, recombinant vector or recombinant cell line comprising the nucleic acid molecule of claim 6.

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