CN114262383A - Epitope polypeptide of helicobacter pylori heat shock protein A and application thereof - Google Patents
Epitope polypeptide of helicobacter pylori heat shock protein A and application thereof Download PDFInfo
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Abstract
The invention discloses an epitope polypeptide of helicobacter pylori heat shock protein A and application thereof. The invention provides a heat shock protein A epitope peptide which is a polypeptide shown in 18 th-28 th position of a sequence 1 or a fusion polypeptide formed by adding a label sequence at the tail end of the polypeptide sequence. The epitope peptide HP19 is searched from the heat shock protein A, and the epitope peptide HP19 can be used for diagnosing or assisting in diagnosing related diseases caused by the infection of the heat shock protein A and the H. The antibody prepared by the epitope peptide can be used for detecting the infection of the heat shock protein A or H.pyrori; provides a basis for further research on the effect of the heat shock protein A in H.pyrori infection immunodiagnosis and immunoprophylaxis.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an epitope polypeptide of helicobacter pylori heat shock protein A and application thereof.
Background
Pyri is a helical, microaerophilic, gram-negative bacterium that colonizes the human stomach. The discovery of the pyhori causes subversive change of understanding of diseases such as chronic gastritis, peptic ulcer and the like in terms of pathogenesis and prevention and treatment. Once obtained, pylori continues to colonize and grow within the human stomach for years, decades, or even a lifetime if untreated. 100% of infected persons develop asymptomatic gastritis, about 30% develop chronic gastritis, 10-20% develop gastric and duodenal peptic ulcers, and about 1% -2% develop gastric adenocarcinoma and gastric mucosa-associated lymphoid tissue lymphoma. Clinical tests show that the detection rate of the H.pyri in chronic gastritis is 54-100%, the detection rate of the H.pyri in chronic active gastritis is more than 90%, the detection rate of the H.pyri in gastric ulcer is more than 80%, and the detection rate of the H.pyri in duodenal ulcer is more than 90%. The incidence of gastric cancer in h.pylori-infected patients is 6-8 times that of non-infected patients, and the incidence of young gastric cancer in h.pylori-infected patients is 20 times that of non-infected patients. In addition to the above diseases, h.pyri has also been shown to be involved in many parenteral diseases, such as those associated with the development of oral, cutaneous, blood, cardiovascular and respiratory systems, and even pregnancy with pediatric diseases.
Therefore, the research on immune response and immune protection mechanism aiming at the H.pyri has positive significance for overcoming the problems existing in the current treatment method, more effectively detecting and evaluating, and even preventing and treating the H.pyri infection.
Pylori heat shock protein a (HspA) is a bacterial heat shock chaperone, one of the key virulence factors and protective antigens of h. HspA consists of 118 amino acids and is divided into two domains: the A domain (1-90 amino acids), which has sequence similarity to the GroES sequence, and the B domain (91-118 amino acids), which is unique to H.pyri and Helicobacter acinonuchi, and contains 8 histidines and 4 cysteines. It is well known that the body resists invasion and pathogenesis of foreign pathogens by recognizing epitopes of pathogens to generate adaptive immune response. Therefore, the identification of the epitope has important significance for immune diagnosis, immune protection mechanism research and vaccine development of infectious diseases.
Disclosure of Invention
The invention aims to provide a heat shock protein A epitope peptide.
The heat shock protein A epitope peptide provided by the invention is a polypeptide shown in 18 th-28 th site of a sequence 1 or a fusion polypeptide formed by adding a tag sequence at the tail end of the polypeptide sequence.
The nucleic acid molecule encoding the heat shock protein A epitope peptide is also within the protection scope of the present invention.
Expression cassettes, recombinant vectors or recombinant cell lines comprising the above-described nucleic acid molecules are also within the scope of the present invention.
Another objective of the invention is to provide a heat shock protein A antigen.
The antigen provided by the invention is formed by coupling the heat shock protein A epitope peptide and a carrier protein.
In the antigen, the carrier protein is keyhole limpet hemocyanin or bovine serum albumin.
The application of the heat shock protein A epitope peptide or the heat shock protein A antigen in preparing the antibody of the heat shock protein A as immunogen is also in the protection scope of the invention.
The application of the nucleic acid molecule for coding the epitope peptide or the expression cassette, the recombinant vector or the recombinant cell line containing the nucleic acid molecule in preparing the anti-heat shock protein A antibody is also within the protection scope of the invention.
The invention also provides the anti-heat shock protein A antibody prepared by the heat shock protein A epitope peptide or the heat shock protein A antigen as immunogen.
It is a further object of the invention to provide a product.
The active component of the product provided by the invention is the heat shock protein A epitope peptide or the heat shock protein A antigen or the antibody;
the product has at least one of the following functions:
(a) for diagnosing or aiding in diagnosing diseases associated with abnormal expression of heat shock protein A;
(b) for preventing diseases related to abnormal expression of heat shock protein A;
(c) for use in the diagnosis or for the aided diagnosis of diseases associated with h.pyri infection;
(d) it can be used for preventing diseases caused by H.pyrori infection.
The use of the above antibody in any of the following is also within the scope of the present invention:
1) detecting or detecting heat shock protein A in an auxiliary way;
2) detecting or aiding in the detection of h.pyri;
3) preparing a product for detecting or assisting in detecting the heat shock protein A;
4) the application of the product in preparation of products for detecting or assisting in detecting related diseases caused by H.
The application of the above-mentioned heat shock protein A epitope peptide or the above-mentioned heat shock protein A antigen or the above-mentioned antibody or the substance using the above-mentioned heat shock protein A 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 the heat shock protein A;
(b) preparing a product for preventing diseases related to abnormal expression of heat shock protein A;
(c) preparing a product for diagnosing or assisting in diagnosing diseases related to H.pyri infection;
(d) preparing a product for preventing diseases related to H.pyrori infection.
The product for preventing the diseases related to the abnormal expression of the heat shock protein A is a vaccine, and particularly can be a multi-epitope vaccine preparation.
The epitope peptide HP19 is screened and identified from the heat shock protein A, can be used for diagnosing or assisting in diagnosing related diseases caused by the infection of the heat shock protein A and the H.pyriri, and can also be used as a vaccine for preventing the related diseases caused by the infection of the H.pyriri. The antibody prepared by the epitope peptide can be used for detecting the infection of the heat shock protein A or H.pyrori; provides a basis for further research on the effect of the heat shock protein A in H.pyrori infection immunodiagnosis and immunoprophylaxis.
Drawings
FIG. 1 shows the screening of the immunodominant peptide fragment of HspA antigen. (A) HspA truncated fragments from h. (B) OD values of five peptides detected by ELISA with 20 mouse anti-rHspA sera.
FIG. 2 shows the fine mapping of the epitope of the HP1 peptide fragment in HspA. (A) Overlapping synthetic peptides covering HP 1. Each peptide fragment comprises 11 amino acids, of which 9 amino acids overlap with adjacent peptide fragments. (B) The ELISA detection results of the 11 polypeptides and the mouse rHspA-resistant serum.
Figure 3 is the immunogenicity and immunoreactivity of HP11 and HP19 epitopes. (A) Antigen epitope-KLH mouse antiserum and synthetic epitope peptide ELISA detection results. Synthetic epitope peptides HP11 and HP19 were used as envelope antigens, epitope-KLH mouse antiserum was used as the primary antibody, and PBS immune mouse serum was used as the negative control. (B) HP11-KLH immune mouse antiserum immunoblot analysis. (C) HP19-KLH immune mouse antiserum immunoblot analysis. (D) Antiserum immunoblot analysis of rHspA immunized mice.
FIG. 4 shows epitope-specific lymphocyte proliferation reactions. The epitope stimulates the splenic lymphocytes of corresponding HP11-KLH (A) and HP19-KLH (B) immunized mice to generate proliferation reaction. The epitope stimulated PBS immunized mice splenic lymphocytes as negative control, ConA stimulation as positive control. Spleen lymphocyte 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 persons. Serum antibody OD values against epitopes HP11 and HP19 were tested by ELISA using 42 HspA seropositive samples from h. Pyloi antibody negative sera were used as negative controls. Positive judgment criteria: OD value > mean OD value of negative control serum 2.1.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Bacterial strains and culture conditions in the following examples:
the h.pyriss2000 sonicate supernatants of the following examples were prepared as follows: h.pylori Strain Sydney Strain 2000(SS2000) (Thompson LJ, Danon SJ, Wilson JE, et al, viral Helicobacter pylori Infection with Sydney Strain 1AND a Newly-Identified Mouse-Adapted Strain (Sydney Strain 2000) in C57BL/6AND BALB/C Mice. INFEON CTIN AND IMMUNITY,2004,72(8): 4668-4679) AND NCTC11637 (American ATCC, ATCC43504) were plated on H.pylori Selective Agar plates (Campybacter Agar Base) containing 7% fetal bovine serum at 37 ℃ with 5% O2、10％CO2And 85% N2Under microaerophilic conditions. After 3 days of culture, colonies were scraped and washed twice with 20mM pre-cooled Phosphate Buffered Saline (PBS) pH7.0, followed by centrifugation at 10,000 Xg for 1 at 4 deg.CBacterial pellets were obtained in 0 minute. The h.pyhori SS2000 pellet was resuspended in PBS, sonicated in an ice bath (300w,10s,10s,20min), centrifuged for 20min, and the h.pyhori SS2000 supernatant, i.e. the HspA-containing supernatant, was collected for Western Blotting (WB). H.pyri NCTC11637 bacteria are precipitated, and the NCTC11637 genome DNA is extracted by using a bacterial genome DNA extraction kit (Beijing Tiangen Biochemical technology Co., Ltd.).
The construction, expression and purification methods of the h.pyrori recombinant hspa (rhspa) and GST fusion peptides in the following examples are as follows:
the gene sequence of HspA (SEQ ID NO: 2) was directly amplified from the genome of helicobacter pylori NCTC11637 by PCR, and cloned into pGEX-6P-1(+) (GE Healthcare) expression vector, placed between BamHI and NotI or XhoI cleavage sites, to obtain a recombinant plasmid. The recombinant plasmid was transformed into E.coli BL21(DE3) pLysS cells (Cwbio) and induced to express the recombinant fusion protein GST-HspA with 1mM IPTG. The cells were harvested by centrifugation and the bacterial pellet resuspended in PBS, the resuspended cells were sonicated, and the supernatant, containing the recombinantly expressed GST-HspA fusion protein, was harvested by centrifugation.
The recombinant GST-HspA fusion protein was purified by Price Glutathione Superflow Agarose (Thermo). Then, GST tag was cleaved with precision protein enzyme (Beyotime), removed using GST tag purification resin (Beyogold), and purified according to the instructions (GE Healthcare, USA)，Obtaining the recombinant protein rHspA.
The recombinant protein rHspA obtained by purification was analyzed by SDS-PAGE, and the concentration of the purified protein was determined by BCA method.
Construction, expression and purification of GST-HP11 and GST-HP19 are similar to GST-HspA fusion proteins except that the gene sequence encoding HspA is replaced with the gene sequence encoding the HP11 or HP19 polypeptide, and the GST tag is not removed.
The synthesis of the HP1-HP5 polypeptide, of the overlapping peptides covering the full length of HP1, and of the HP11 polypeptide coupled to the HP19 Keyhole Limpet Hemocyanin (KLH) (either HP11-KLH coupled peptide or HP19-KLH coupled peptide was prepared) in the examples described below were performed with GenScript (Nanjing, China). Standard solid phase FMOC method was used for peptide synthesis with > 95% purity assessed by high pressure liquid chromatography and identified by laser desorption mass spectrometry. The peptides were dissolved in DMSO or ultrapure water at a concentration of 10mg/ml and stored in portions at-20 ℃.
The ELISA detection method in the following examples was as follows:
for coating with polypeptide as antigen, 96-well ELISA plates (Costar) were pretreated with 150. mu.l of 2.5% glutaraldehyde for 1 hour at 37 ℃ and then washed four times with water. Epitope peptide as coating antigen was diluted in 0.1mM carbonate buffer (pH9.6) and coated on ELISA plates (30. mu.g/ml, 100. mu.l/well) and incubated overnight at 37 ℃. When HspA was coated with the antigen, the antigen was diluted in 0.1mM carbonate buffer (pH9.6) and coated on an ELISA plate (2. mu.g/ml, 100. mu.l/well), and incubated overnight at 4 ℃. Wash the plate 3 times with PBST.
Blocking buffer (PBST containing 5% skim milk for mouse serum, protein-free blocking solution (Thermo) for human serum) was added to the ELISA plates at 200. mu.l/well and blocked at 37 ℃ for 1 hour. Blocking solution was removed and 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 at 37 ℃ for 1 hour. PBS immunized mice or h.pyri infection negative healthy human serum were used as negative controls.
After washing the 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 antibody was added to the wells (100. mu.l/well), incubated at 37 ℃ for 1 hour, and the ELISA plates were washed as above. The enzyme substrate solution 3,3,5, 5-tetramethylbenzene-biphenyl (TMB) was added at 100. mu.l/well, and the reaction was carried out at room temperature for 15 minutes. Using 100. mu.l of 1MH2SO4The reaction was terminated and the absorbance at 450nm (A450) was measured using an ELISA plate reader. All assays were performed in duplicate wells, and the assay results were averaged in duplicate wells.
The rhspA antiserum in the following examples was prepared by immunizing female SPF BALB/c mice (20 per group, Central laboratory of military medicine), 6-8 weeks old, and the primary immunization was performed subcutaneously using an emulsion of 50. mu.g of rhspA mixed with complete Freund's adjuvant (Sigma). On days 10 and 20 post-immunization booster immunizations were performed with 50 μ g rHspA mixed with incomplete freund's adjuvant (Sigma). Mice were sacrificed on day 30 and serum samples were collected as anti-rHspA sera.
Statistical analysis in the examples below, all data are expressed as mean ± standard deviation (s.d.). Mean values were compared using a two-tailed t-test, and analysis using GraphPad prism8.0.2(GraphPad Software) was considered statistically significant for P < 0.05.
Example 1 identification and preparation of epitope peptide of Heat shock protein A (HspA) and detection of immunogenicity and immunoreactivity
First, screening of HspA antigen immunodominance reaction peptide
Cutting HspA (the amino acid sequence is shown as sequence 1 in a sequence table) into five segments, wherein the first 4 peptide segments are positioned in an A structural domain of the HspA, each segment is overlapped with 8 amino acids, and the segments are sequentially named as HP1-HP4 from the N end to the C end; the 5 th peptide stretch is located in the B domain, overlapping HP4 by 2 amino acids (fig. 1A).
The amino acid sequence of the HP1 peptide segment is 2 nd to 31 th of the sequence 1;
the amino acid sequence of the HP2 peptide segment is 34 th to 53 th of the sequence 1;
the amino acid sequence of the HP3 peptide segment is 46 th to 75 th of the sequence 1;
the amino acid sequence of the HP4 peptide segment is 68 th-91 th position of the sequence 1;
the amino acid sequence of the HP5 peptide segment is the 90 th to 118 th position of the sequence 1;
the above five polypeptides were synthesized as antigens and detected by ELISA using 20 mouse anti-rHspA sera (HspA antiserum).
The results are shown in fig. 1B, which indicates that HP1 reacts with all antisera with the strongest immune response, and HP1 is the HspA antigen immunodominant reaction segment.
II, fine positioning of HP1 epitope
To fine-localize 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, resulting in a total of 11 polypeptides (fig. 2A).
The amino acid sequence of the HP11 polypeptide is 2 nd to 12 th of the sequence 1;
the amino acid sequence of the HP12 polypeptide is the 4 th-14 th position of the sequence 1;
the amino acid sequence of the HP13 polypeptide is 6 th to 16 th of the sequence 1;
the amino acid sequence of the HP14 polypeptide is the 8 th to the 18 th positions of the sequence 1;
the amino acid sequence of the HP15 polypeptide is 10 th-20 th of the sequence 1;
the amino acid sequence of the HP16 polypeptide is 12 th to 22 th 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 16 th to 26 th of the sequence 1;
the amino acid sequence of the HP19 polypeptide is 18 th to 28 th of the sequence 1;
the amino acid sequence of the HP20 polypeptide is 20 th to 30 th of the sequence 1;
the amino acid sequence of the HP21 polypeptide is 21 st to 31 st of the sequence 1;
the polypeptides were synthesized, ELISA plates were coated with the synthesized polypeptides as coating antigens, and the reactivity of 11 polypeptides with 20 mouse anti-rHspA sera (HspA antiserum) was examined by ELISA.
As a result, as shown in fig. 2B, it can be seen that HP11, HP18, HP19 and HP20 are able to generate stronger immune responses. Among them, the sequences of three peptides HP18, HP19 and HP20 are similar, and HP19(ENKTSSGIIIP) has the strongest immune response, and HP19 is presumed to be an epitope of HspA. In addition, the immune response of HP11(KFQPLGERVLV) was comparable to HP20, possibly being another epitope of HspA.
Immunogenicity and immunoreactivity detection of HP11 epitope peptide and HP19 epitope peptide
In preliminary identification of two B-cell epitopes in HspA, it was first assessed whether these epitopes could induce an immune response in mice, as follows:
1. KLH-coupled peptide of HP11 (HP11-KLH) and KLH-coupled peptide of HP19 (HP19-KLH)
To enhance the immunogenicity of the polypeptides, a cysteine was attached to the C-terminus of HP11 and HP19, which were then conjugated to KLH using the MBS method to form KLH-conjugated peptides.
The HP11-KLH coupling peptide is a polypeptide obtained by connecting a cysteine to the C terminal of HP11, and then the HP11 connected with the cysteine to the C terminal is coupled to the KLH to form the HP11-KLH coupling peptide (the charging mass ratio of the HP11 connected with the cysteine to the C terminal to the KLH is 1: 1).
The HP19-KLH coupling peptide is a polypeptide obtained by connecting a cysteine to the C terminal of HP19, and then the HP19 connected with the cysteine to the C terminal is coupled to the KLH to form the HP19-KLH coupling peptide (the charging mass ratio of the HP11 connected with the cysteine to the C terminal to the KLH is 1: 1).
2. Mouse immunization and sample collection
Immunogenicity of rHspA and HP11-KLH conjugated peptide or HP19-KLH conjugated peptide was assessed by immunizing female SPF BALB/c mice (10-20 per group, central laboratory animals of the military medical institute) at 6-8 weeks of age.
Primary immunization was performed subcutaneously using 50. mu.g of rHspA or 50. mu.g of HP11-KLH conjugated peptide or 50. mu.g of HP19-KLH conjugated peptide, respectively, in emulsion mixed with complete Freund's adjuvant (Sigma).
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 primary immunization with incomplete Freund's adjuvant (Sigma), respectively.
Control mice were immunized with PBS using the same immunization protocol.
The mice were sacrificed on day 30 and serum samples were collected to give rHspA immune serum, HP 11-KLH-conjugated peptide immune serum, HP 19-KLH-conjugated peptide immune serum and analyzed by ELISA and western blot; collecting spleen tissues to obtain rHspA immune mouse spleen cells, HP11-KLH coupling peptide immune mouse spleen cells and HP19-KLH coupling peptide immune mouse spleen cells to perform lymphocyte proliferation tests.
All animals were purchased from vindolite (beijing, china) and raised under SPF conditions.
3. ELISA for detecting immunogenicity of antigen epitope
According to the method described above, the mouse HP11-KLH conjugated peptide immune serum (1:100, also called HP11-KLH antiserum) and HP19-KLH conjugated peptide immune serum (1:100, also called HP11-KLH antiserum) collected from 2 above were used as antibodies, and all sera obtained from mice immunized with KLH conjugated peptide were tested by ELISA. Sera from mice immunized with PBS served as negative controls.
The detection result is shown in fig. 3A, the injection of HP11-KLH conjugate peptide and HP19-KLH conjugate peptide can induce mice to generate strong immune response, and the titer of 2 epitope peptide specific antibodies (antiserum) exceeds 1: 1000. both epitopes were able to produce a positive ELISA reaction with sera from mice immunized with KLH-conjugated peptide and neither epitope with sera from mice immunized with KLH-conjugated peptide or PBS (fig. 3A).
4. Immune reactivity of antigen epitope detected by immune blotting analysis method
Western immunoblot analysis of sera immunized with KLH-coupled peptide with h.pylori SS2000 supernatant, rHspA and GST fusion proteins was performed as follows:
supernatant of the ultrasonication of H.pyri SS2000 was prepared according to the above method;
the amino acid sequence of the rHspA protein is the protein of sequence 1.
The amino acid sequence of the GST fusion expression protein GST-HP11 is sequence 3, wherein the 1 st to 229 th positions of the sequence 3 are GST tags, the 230 nd to 231 th positions of the sequences are linkers, and the 232 nd to 242 th positions of the sequences are HP 11.
The amino acid sequence of the GST fusion expression protein GST-HP19 is sequence 4, wherein the 1 st to 229 th positions of the sequence 4 are GST tags, the 230 nd to 231 th positions of the sequences are linkers, and the 232 nd to 242 th positions of the sequences are HP 19.
The rHspA protein is used as a positive control, and the GST protein is used as a negative control.
Supernatant of H.pyrori SS2000 sonicate (SS2000), 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 then blocked with blocking buffer (5% skim milk in PBST, pH7.4)) at 37 ℃ for 1 hour to prevent non-specific protein binding. The membrane was incubated with mouse HP11-KLH conjugated peptide immune serum (1:500 dilution) or HP19-KLH conjugated peptide immune serum (1:500 dilution) collected from 2 above at 37 ℃ for 1 hour and washed 3 times with PBST at room temperature. HRP-conjugated rabbit anti-mouse IgG (1:5,000 dilution, Abcam) was used at 37 ℃ for 1 hour and detected using the HRP Western blot assay kit (Easybio, China).
As shown in FIGS. 3B and 3C, the HP11-KLH immune serum (HP11-KLH antiserum) and HP19-KLH immune serum (HP19-KLH antiserum) were immunoblotted against native HspA (SS2000) of H.pylori SS2000, rHspA expressed in E.coli and GST fusion proteins themselves (GST-HP11 or GST-HP19), but not GST (FIGS. 3B and 3C).
Subsequently, antisera prepared by immunizing mice with rhspA were immunoblotted with native Hspa from SS2000, rhspA protein, and two GST fusion proteins GST-HP11 and GST-HP 19. The results confirmed that both fusion proteins GST-HP11 and GST-HP19, as well as SS2000 native HspA and rHspA proteins, were immunoblotted against the rHspA antiserum, whereas GST alone did not react with the rHspA antiserum (FIG. 3D).
5. Lymphocyte proliferation response assay
To further understand the immune response induced by the epitope, the stimulation of specific lymphocyte proliferation activity by the epitope from splenic lymphocytes of mice immunized with the epitope-conjugated peptide KLH was examined. Spleen cells of mice immunized with HP11-KLH or HP19-KLH were stimulated with the corresponding epitopes, while spleen cells of mice immunized with PBS were stimulated with the same epitopes as a negative control, and concanavalin A (ConA, Solarbio, cat # C8110) as a positive control. The method comprises the following specific steps:
the spleen tissue of mice immunized with the HP 11-KLH-conjugated peptide prepared in 2 above, the spleen tissue of mice immunized with the HP 19-KLH-conjugated peptide, and the nylon mesh were gently pressed using a syringe stopper to prepare single cell suspensions. Cells were seeded in 96-well cell culture plates in RPMI-1640 medium (Gibco, USA) containing 10% FBS at 4X 10/well5And (4) cells. Under the condition of stimulation without any stimulator (negative control) and with 10. mu.g/mL epitope peptide HP11 polypeptide or HP19 polypeptide or 0.625. mu.g/mLCona (corresponding concentration of epitope peptide or ConA is added to the cells), 5% CO at 37 ℃. (negative control)2Cultured under the conditions for 72 hours. Mice immunized with PBS served as negative control group (negative control). During the last four hours of incubation, 20. mu.l of Cell Counting Kit-8(CCK8,dojindo, japan) and a450 was measured using SpectraMaxi3x (Molecular Devices, usa). Spleen lymphocyte Stimulation Index (SI) is the OD of the stimulated wells divided by the OD of the control wells.
As shown in FIG. 4, ConA can stimulate the splenocytes of mice to generate nonspecific proliferation, and the epitope peptide stimulates the splenic lymphocyte proliferation activity of the corresponding epitope peptide-KLH coupled peptide immunized mice to be significantly higher than that of the negative control group (P <0.01, FIG. 4).
Fourth, antibody detection of natural infector antigen epitope
The antibody expression profiles of the two B-cell epitopes identified in HspA were examined with sera from naturally infected h.
The study was approved by the ethical committee of the fourth medical center of the general hospital of the liberty and informed consent was obtained from the patients. Hospitalized patient sera were collected and tested for the presence of anti-h.pyri antibodies using the h.pyri IgG ELISA kit (IBL). The experimental procedures were performed according to the kit instructions. Sera were considered to be h.pyri positive when the cutoff index (COI) >1.2 and h.pyri negative when the cutoff index (COI) < 0.8.
85 H.pyri positive serums (named as P1-P85) are selected to detect HspA antibody and epitope antibody by an ELISA method, the envelope antigens are respectively rHspA, epitope peptides HP11 and HP19, and 17 H.pyri antibody negative serums are selected as negative controls. The positive judgment limit of HspA antibody and epitope antibody is: OD value > mean OD value of negative control serum 2.1.
The results are as follows: among 85 h.pyri infected patients, 42 HspA antibodies were detected positive by ELISA, with a positive rate of 49.4%. Pylori IgG ELISA kits tested negative sera (N1-N17) as negative controls. Of 42 HspA seropositive patients, 9 were positive for the HP11 epitope and 14 were positive for the HP19 epitope, with a positive rate of 21.4% and 33.3%, respectively. The lower panel shows the antibody response spectrum of two epitopes in HspA seropositive patients (fig. 5).
SEQUENCE LISTING
<110> military medical research institute of military science institute of people's liberation force of China
<120> epitope polypeptide of helicobacter pylori heat shock protein A and application thereof
<160> 4
<170> PatentIn version 3.5
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Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
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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 (10)
1. The heat shock protein A epitope peptide is a polypeptide shown in 18 th to 28 th positions of a sequence 1 or a fusion polypeptide formed by adding a tag sequence at the tail end of the polypeptide sequence.
2. A nucleic acid molecule encoding the heat shock protein A epitope peptide of claim 1.
3. An expression cassette, recombinant vector or recombinant cell line comprising the nucleic acid molecule of claim 2.
4. The heat shock protein A antigen is prepared by coupling the heat shock protein A epitope peptide of claim 1 with a carrier protein.
5. The heat shock protein A antigen of claim 4, wherein: the carrier protein is keyhole limpet hemocyanin or bovine serum albumin.
6. Use of a heat shock protein a epitope peptide of claim 1 or a heat shock protein a antigen of claim 4 or 5 as an immunogen for the preparation of an antibody against heat shock protein a;
or, the use of the nucleic acid molecule of claim 2 or the expression cassette, recombinant vector or recombinant cell line of claim 3 for the preparation of an antibody against heat shock protein a.
7. An antibody against heat shock protein A prepared from the heat shock protein A epitope peptide of claim 1 or the heat shock protein A antigen of claim 4 or 5 as an immunogen.
8. A product, the active ingredient of which is the heat shock protein a epitope peptide of claim 1 or the heat shock protein a antigen of claim 2 or 3 or the antibody of claim 7;
the product has at least one of the following functions:
(a) for diagnosing or aiding in diagnosing diseases associated with abnormal expression of heat shock protein A;
(b) for preventing diseases related to abnormal expression of heat shock protein A;
(c) for use in the diagnosis or for the aided diagnosis of diseases associated with h.pyri infection;
(d) it can be used for preventing diseases caused by H.pyrori infection.
9. Use of the antibody of claim 7 in any one of:
1) detecting or detecting heat shock protein A in an auxiliary way;
2) detecting or aiding in the detection of h.pyri;
3) preparing a product for detecting or assisting in detecting the heat shock protein A;
4) the application of the product in preparation of products for detecting or assisting in detecting related diseases caused by H.
10. The use of the heat shock protein A epitope peptide of claim 1, or the heat shock protein A antigen of claim 4 or 5, or the antibody of claim 7, or a substance comprising the heat shock protein A epitope peptide of claim 1 as an active ingredient in any one of the following (a) to (d):
(a) preparing a product for diagnosing or assisting in diagnosing diseases related to abnormal expression of the heat shock protein A;
(b) preparing a product for preventing diseases related to abnormal expression of heat shock protein A;
(c) preparing a product for diagnosing or assisting in diagnosing diseases related to H.pyri infection;
(d) preparing a product for preventing diseases related to H.pyrori infection.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1887349A (en) * | 2006-07-20 | 2007-01-03 | 中国人民解放军第三军医大学 | Helicobacter pylori vaccine based on urease B subunit active segment and its prepn process |
| CN101538550A (en) * | 2009-04-30 | 2009-09-23 | 中国人民解放军军事医学科学院生物工程研究所 | Helicobacter pylori viable bacteria carrier vaccine and special recombination bacteria thereof |
| CN101863963A (en) * | 2010-05-21 | 2010-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Helicobacter pylori antigen epitope polypeptide and application thereof |
| CN101863965A (en) * | 2010-05-21 | 2010-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Helicobacter pylori urease B antigenic epitope polypeptide and application thereof |
| CN101863964A (en) * | 2010-05-21 | 2010-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Helicobacter pylori urease B antigen epitope polypeptide and application thereof |
-
2021
- 2021-12-24 CN CN202111598516.7A patent/CN114262383B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1887349A (en) * | 2006-07-20 | 2007-01-03 | 中国人民解放军第三军医大学 | Helicobacter pylori vaccine based on urease B subunit active segment and its prepn process |
| CN101538550A (en) * | 2009-04-30 | 2009-09-23 | 中国人民解放军军事医学科学院生物工程研究所 | Helicobacter pylori viable bacteria carrier vaccine and special recombination bacteria thereof |
| CN101863963A (en) * | 2010-05-21 | 2010-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Helicobacter pylori antigen epitope polypeptide and application thereof |
| CN101863965A (en) * | 2010-05-21 | 2010-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Helicobacter pylori urease B antigenic epitope polypeptide and application thereof |
| CN101863964A (en) * | 2010-05-21 | 2010-10-20 | 中国人民解放军军事医学科学院生物工程研究所 | Helicobacter pylori urease B antigen epitope polypeptide and application thereof |
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| CN114262383B (en) | 2023-06-27 |
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