CN115980345A - Method for detecting helicobacter pylori and application - Google Patents
Method for detecting helicobacter pylori and application Download PDFInfo
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- CN115980345A CN115980345A CN202211046382.2A CN202211046382A CN115980345A CN 115980345 A CN115980345 A CN 115980345A CN 202211046382 A CN202211046382 A CN 202211046382A CN 115980345 A CN115980345 A CN 115980345A
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Abstract
The invention relates to the technical field of helicobacter pylori detection, and particularly discloses a method for detecting helicobacter pylori and application thereof. The method of the invention uses the antigen protein with the amino acid sequence shown as SEQ ID No.1 or the antigen peptide shown as SEQ ID No.2-5 as a target spot to detect the helicobacter pylori in the sample to be detected. Provides a new effective mode for detecting the helicobacter pylori.
Description
Technical Field
The invention relates to the technical field of helicobacter pylori detection, in particular to a method for detecting helicobacter pylori and application thereof.
Background
Helicobacter pylori (Helicobacter pylori) is a gram-negative, spiral microaerophilic bacterium. The pylori penetrates into a thick mucous layer on the surface of a gastric mucosa through flagella movement to reach the surface of epithelial cells of the gastric mucosa, and is planted on the surface of the cells by virtue of pili or adhesin on the surfaces of thalli, so that a host defense mechanism is overcome, the pylori grows and breeds, and a series of pathophysiological injuries are generated to organisms. Many people infected with h.pyri have no symptoms or complications, but all infected people have chronic active gastritis, peptic ulcer occurs in about 15% to 20%, h.pyri-related dyspepsia occurs in 5% to 10%, and gastric malignant tumor (gastric cancer, MALT lymphoma) occurs in 1%.
In view of the high correlation between h.pyri infection and gastric carcinogenesis, as well as the ubiquitous presence of chronic active gastritis and other potential risks following infection, clinical experts are now in consensus with recommendations to suggest drug eradication therapy whenever there is infection, whether symptoms are caused following h.pyri infection, and whether symptoms are mild or severe, whereas diagnostic detection of h.pyri infection is a prerequisite for therapy.
The detection of the pylori can be mainly divided into two types, one type is invasive detection, and detection and diagnosis are carried out by means of histology, bacterial culture, rapid Urease Test (RUT), helicobacter pylori gene detection and the like depending on a tissue specimen obtained by gastroscopy; another class of non-invasive assays, consisting essentially of 13 C or 14 The kit comprises a C Urea Breath Test (UBT), serological examination, helicobacter pylori fecal antigen detection and the like, wherein the detection does not need gastroscopy, avoids pain and possible complications in the process of taking tissues, is convenient to operate, and is widely applied to large-scale crowd screening and clinical diagnosis. Urea breath test is the gold standard of current testing, but it requires special equipment and patients need to discontinue PPI 2 weeks prior to the test or antimicrobial drugs 4 weeks prior to the test, while the presence of other urokinase-producing pathogens in the stomach may lead to false positive results; serological examination has limited application because the current disease and the past infection cannot be distinguished; the detection of helicobacter pylori fecal antigen is continuously favored in recent years due to the advantages of convenient operation, no dependence on equipment, home self-test and the like, although the sensitivity and the specificity of partial products on the market are equivalent to the results of the breath test in scientific research, the large-scale crowd test data support is still lacked, and the detection can only be used as the supplement of the breath test at present and cannot be used for the supplement of the breath testCompletely replace the breath test. The development of a fecal antigen detection kit with high sensitivity and high specificity requires that one or more good antigens which can be used as detection targets must be selected, and the antigens have high protein abundance, sequence conservation and high homology in most or all H.pyri strains, and have low similarity with homologous protein sequences in other intestinal flora so as to ensure the detection accuracy. Therefore, the identification of specific helicobacter pylori antigens which can be used for detection has important practical application value.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, the present invention provides a novel method for effectively detecting helicobacter pylori.
In order to achieve the purpose, the technical scheme of the invention is as follows:
a method for detecting helicobacter pylori takes an antigen protein with an amino acid sequence shown as SEQ ID No.1 or an antigen peptide shown as SEQ ID No.2-5 as a target spot to detect the helicobacter pylori in a sample to be detected.
The antigenic peptide shown in SEQ ID No.2-5 is a B cell linear epitope on the antigenic protein shown in SEQ ID No. 1.
SEQ ID No.2：GTDFFTVHKA；
SEQ ID No.3：EEIYEEFAD；
SEQ ID No.4：VQLGHHPLVTLSEA；
SEQ ID No.5：TRVKEETKTSF。
The research of the invention finds that the antigen protein shown in SEQ ID No.1 has good conservation in helicobacter pylori, has low homology with other bacteria, and can realize effective specificity detection of the helicobacter pylori by taking the antigen protein as a target spot.
The invention also provides a method for detecting helicobacter pylori, which takes the antigen protein with the amino acid sequence shown as SEQ ID No.1 and/or the antigen peptide shown as SEQ ID No.2-5 as a target spot and detects the helicobacter pylori in a sample to be detected by a non-antigen antibody reaction method.
In the method of the present invention, the non-antigen antibody reaction method is mass spectrometry.
In the detection of the actual antigen protein or antigen peptide, an antigen antibody reaction method can be adopted, and other non-antigen antibody reaction methods known in the art can also be utilized, such as mass spectrometry or other methods which can directly identify the antigen protein shown in SEQ ID No.1 and/or the antigen peptide shown in SEQ ID Nos. 2-5.
By applying mass spectrometry, the protein antigen discovered by the invention is used as a target spot to detect helicobacter pylori, whether helicobacter pylori exists in a sample can be identified directly through a characteristic spectrogram of the protein, and whether helicobacter pylori exists in the sample can also be identified through a polypeptide characteristic spectrogram after the protein is digested by trypsin.
The invention also provides a method for detecting the helicobacter pylori, which utilizes a polyclonal antibody and/or a monoclonal antibody to detect the helicobacter pylori in a sample to be detected, wherein the polyclonal antibody is prepared from a plurality of antigen proteins shown in SEQ ID No.1 or antigen peptides shown in SEQ ID Nos. 2-5; the monoclonal antibody is prepared from any one of the antigenic peptides shown in SEQ ID No. 2-5.
The preparation of antibodies for detecting the target can be carried out by those skilled in the art by combining the above-mentioned antigen protein or antigen peptide found by the present invention with the conventional techniques in the art.
The protein antigen or antigen peptide found by the invention is used as a target spot to detect helicobacter pylori by using an antigen-antibody method, including but not limited to an enzyme-linked immunosorbent assay (Elisa), an immunoblotting method (WB) and a colloidal gold method, and the specific detection method is disclosed and mature in the field and is not described again.
In the method of the invention, before the detection of the sample to be detected, the steps of screening and enriching helicobacter pylori can be further included.
The invention also provides a method for screening and enriching the helicobacter pylori, which takes the antigen protein with the amino acid sequence shown as SEQ ID No.1 or the antigen peptide shown as SEQ ID No.2-5 as a target spot to screen and enrich the helicobacter pylori in a sample to be detected.
In the method of the present invention, the screening and enrichment can be achieved by coupling magnetic beads with polyclonal antibodies and/or monoclonal antibodies against the target of the present invention, or can be performed by a conventional method known in the art, which is not limited by the present invention.
In the method of the present invention, the sample to be tested is saliva, stool, gastric mucus, gastric tissue slice or blood.
The detection methods of the present invention are all not directly used for disease diagnosis or treatment purposes, and can be used for detecting/monitoring whether helicobacter pylori and specific components thereof exist in the environment or in a sample.
The invention also provides the application of the antigen protein or the antigen peptide in the preparation of a reagent or a kit for diagnosing and detecting helicobacter pylori, wherein the amino acid sequence of the antigen protein is shown as SEQ ID No.1, and the amino acid sequence of the antigen peptide is shown as SEQ ID No. 2-5.
The invention also provides a reagent or a kit for detecting helicobacter pylori, which comprises a polyclonal antibody and/or a monoclonal antibody, wherein the polyclonal antibody is prepared from a plurality of antigen proteins shown in SEQ ID No.1 or antigen peptides shown in SEQ ID Nos. 2-5; the monoclonal antibody is prepared from any one of the antigenic peptides shown in SEQ ID No. 2-5.
The invention has the beneficial effects that:
the invention provides a helicobacter pylori detection method with good coverage and strong specificity, and provides a new effective way for detecting helicobacter pylori.
Drawings
FIG. 1 shows the WB detection results of the present invention.
FIG. 2 shows the result of SDS-PAGE protein electrophoresis, in which the positions of the gel-cutting bands are indicated by boxes.
The results of the sequence identity alignment in h.
Fig. 4 is a sequence identity alignment of h.
Detailed Description
Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
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 or can be prepared by a method conventional in the art unless otherwise specified.
Example 1h. Pyrori strain culture
9 H.pylor strains (derived from the China helicobacter pylori strain bank of China center for disease prevention and control, with the numbers of CHPC 1.9799, CHPC 1.9800, CHPC 1.9801, CHPC 1.9802, CHPC 1.9803, CHPC 1.9804, CHPC 1.9805, CHPC 1.9806, CHPC 1.9807) stored at-80 ℃ were rescued, smeared on Columbia plates containing 5% sheep blood, and subjected to microaerophilic conditions (5% O) 2 ,10％ CO 2 ,85％ N 2 ) Incubated at 37 ℃ for 48 hours. H.pyri was positively identified by colony morphology and catalase, oxidase and urease.
Example 2h. pyrori inactivated vaccine preparation and chicken immunization
Scraping the above strains cultured for 48 hr in 0.9% physiological saline to obtain 10 9 CFU/ml mixed bacterial liquid, then in 0.6% formaldehyde solution for 24 hours after inactivation preparation emulsion vaccine (complete Freund's adjuvant, with bacterial liquid 1:1 mixture) for chicken immunization.
9 chickens are immunized by each strain for four times, each chicken is immunized by 0.5ml for the first time, and intramuscular injection is carried out at two points; immunizing 1ml of each chicken for the second time, and performing subcutaneous injection at three points; immunizing 1.5ml of each chicken for the third time, and performing subcutaneous injection at three points; 1.5ml of each chicken is immunized in a fourth time, and subcutaneous injection is carried out at three points, and the interval time of each immunization is two weeks.
EXAMPLE 3 preparation of yolk antibody IgY
Collecting eggs after 4 times of immunization, wiping and disinfecting eggshells, beating eggs by adopting a manual machine, removing egg white, puncturing a yolk membrane, and collecting yolk. The volume of the yolk solution was measured, and then added with ultrapure water 1 (yolk: double distilled water) having a pH of 5.0 at a ratio of 10, stirred well at 4 ℃ and incubated overnight (16 hours). 3000g, centrifuging at 4 deg.C for 1h, adding equal volume of saturated ammonium sulfate into the supernatant to 50% saturation, incubating at 4 deg.C for 1h,8000g, centrifuging at 4 deg.C for 30min, resuspending the precipitate with ultrapure water to original volume, adding 19% sodium sulfate (190 g/L), and incubating at room temperature for 20min.8000g, centrifuging at 25 ℃ for 20min, resuspending the precipitate to the original volume with ultrapure water, adding 14% sodium sulfate (140 g/L), incubating at normal temperature for 20min,8000g, centrifuging at 25 ℃ for 20min, freeze-drying the precipitate to form dry powder, thus obtaining the anti-helicobacter pylori yolk antibody dry powder, and storing at-20 ℃ for later use.
Example 4WB assay for detection of reaction between IgY and H
A bacterial solution (10) of 9 strains of the strain obtained in example 1 was collected 8 CFU/mL), 10. Mu.l each was subjected to SDS-PAGE protein electrophoresis after adding a Loading buffer and boiling for 10min (see FIG. 2). After electrophoresis is finished, membrane transfer is performed. The membrane-transferring conditions of the wet-transfer electrophoresis apparatus are as follows: 350mA,30min. Washing the membrane with Tris Buffer (TBST) containing 0.1% Tween20 for 3 times, 10min each time; adding blocking solution (1 × TBST +5% skimmed milk powder), blocking at room temperature for 3 hr, washing membrane with TBST for 10min for 3 times; the chicken IgY obtained in example 3, diluted in 1. Washing the membrane for 3 times by TBST, 10min each time, adding a secondary antibody reaction solution (horseradish peroxidase-labeled goat anti-chicken IgY secondary antibody) of 1. Adding color developing liquid for developing.
Example 5 identification of consensus dominant antigens
The WB analysis result (figure 1) shows that the different strains have good immune effect after being immunized, the IgY has better reaction with self-strain antigen WB and better cross reaction with other strains, and a plurality of bands which are universally existed in the different strains and better react with the IgY of each group are found, and a plurality of protein antigens which are universally existed in H.pyrori and better generate by the IgY after being immunized into chickens are obtained by cutting gel from the positions of SDS-PAGE gel blocks (the gel cutting positions are marked by boxes in figure 2), performing in-gel enzymolysis and identifying by mass spectrum.
Example 6 antigen selection for detection/diagnosis of helicobacter pylori
One of the proteins obtained in example 5, corresponding to between 18 and 25kD on SDS-PAGE (corresponding to the bottom band in FIG. 1), was further analyzed and its amino acid sequence is shown in SEQ ID No. 1. Performing blastp alignment on the sequences in H.pyri to find that the sequences exist in all 1000 H.pyri strains participating in alignment, extracting the aligned sequences to perform multi-sequence alignment, and finding that the sequences have good conservation in H.pyri and homology of more than 95% (see figure 3); after the aligned species excluded helicobacter, blastp alignments were performed and the first 100 positions with the highest homology were found to be between 45% and 53% (see fig. 4). Therefore, the protein is used as a detection target of H.pyri, and has good strain coverage and specificity.
Example 7 prediction of epitope peptides
The protein is predicted by using an online B cell Linear Epitope Prediction tool Bepipred Linear Epitope Prediction 2.0 (http:// tools. Iedb. Org/bcell /), and the sequences SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5 are found to be sequences with immunogenicity.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (9)
1. A method for detecting helicobacter pylori is characterized in that antigen protein with an amino acid sequence shown as SEQ ID No.1 or antigen peptide shown as SEQ ID No.2-5 is taken as a target spot to detect the helicobacter pylori in a sample to be detected.
2. A method for detecting helicobacter pylori, utilizes polyclonal antibody and/or monoclonal antibody to detect helicobacter pylori in a sample to be detected, and is characterized in that the polyclonal antibody is prepared from a plurality of antigen proteins shown in SEQ ID No.1 or antigen peptides shown in SEQ ID Nos. 2-5; the monoclonal antibody is prepared from any one of the antigenic peptides shown in SEQ ID No. 2-5.
3. A method for detecting helicobacter pylori is characterized in that the detection of the helicobacter pylori in a sample to be detected is carried out by a non-antigen antibody reaction method by taking an antigen protein with an amino acid sequence shown as SEQ ID No.1 and/or an antigen peptide shown as SEQ ID No.2-5 as a target spot.
4. The method of claim 3, wherein the non-antigen antibody reaction method is mass spectrometry.
5. The method as claimed in any one of claims 1 to 4, wherein the step of screening and enriching H.pylori is further included before the detection of the sample to be tested.
6. A method for screening and enriching helicobacter pylori is characterized in that the screening and enrichment of the helicobacter pylori in a sample to be detected is carried out by taking an antigen protein with an amino acid sequence shown as SEQ ID No.1 or an antigen peptide shown as SEQ ID No.2-5 as a target spot.
7. The method according to any one of claims 1 to 6, wherein the sample to be tested is saliva, stool, gastric mucus, gastric tissue slices or blood.
8. The antigen protein or the antigen peptide is applied to the preparation of a reagent or a kit for diagnosing and detecting helicobacter pylori, the amino acid sequence of the antigen protein is shown as SEQ ID No.1, and the amino acid sequence of the antigen peptide is shown as SEQ ID No. 2-5.
9. A reagent or a kit for detecting helicobacter pylori comprises a polyclonal antibody and/or a monoclonal antibody, and is characterized in that the polyclonal antibody is prepared from a plurality of antigen proteins shown in SEQ ID No.1 or antigen peptides shown in SEQ ID Nos. 2-5; the monoclonal antibody is prepared from any one of the antigenic peptides shown in SEQ ID No. 2-5.
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