CN114480356A - Helicobacter pylori monoclonal antibody and application thereof - Google Patents

Helicobacter pylori monoclonal antibody and application thereof Download PDF

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CN114480356A
CN114480356A CN202210286040.1A CN202210286040A CN114480356A CN 114480356 A CN114480356 A CN 114480356A CN 202210286040 A CN202210286040 A CN 202210286040A CN 114480356 A CN114480356 A CN 114480356A
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helicobacter pylori
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钟小强
段超
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Abstract

The invention discloses a helicobacter pylori UreB antigen epitope polypeptide and a monoclonal antibody for resisting the epitope, wherein the nucleotide sequences of a heavy chain variable region and a light chain variable region of the monoclonal antibody are shown as SEQ ID NO.1 and SEQ ID NO. 2. The invention also discloses a kit for detecting helicobacter pylori antibody, which comprises an effective amount of the epitope polypeptide of claim 1, the monoclonal antibody of claim 2 and a matched detection reagent. The epitope polypeptide prepared by the invention has good immunogenicity and specificity; the monoclonal antibody prepared by the invention has good specificity and sensitivity; the diagnostic kit prepared by the epitope polypeptide and the monoclonal antibody has good specificity, sensitivity and accuracy, and is suitable for large-scale application.

Description

Helicobacter pylori monoclonal antibody and application thereof
Technical Field
The invention belongs to the technical field of epidemic disease diagnosis, and particularly relates to a helicobacter pylori monoclonal antibody and application thereof.
Background
Helicobacter pylori (Hp) is a bacterium that is helicoidal, microaerobic, and has very stringent growth conditions. The first successful isolation from biopsy tissue from the gastric mucosa of patients with chronic active gastritis in 1983 is the only microorganism species currently known to survive in the human stomach. In 2017, 10 and 27, the list of carcinogens published by the international cancer research institution of the world health organization is preliminarily collated for reference, and helicobacter pylori (infection) is in a list of carcinogens. The helicobacter pylori diseases include gastritis, peptic ulcer, lymphoproliferative gastric lymphoma, etc. caused by infection with helicobacter pylori. An adverse consequence of H.pylori disease is gastric cancer.
Helicobacter pylori is a gram-negative bacterium, which is mainly distributed in gastric mucosal tissues of animals such as macaque, rat, pig, dog and the like, and 67-80% of gastric ulcer and 95% of duodenal ulcer are caused by helicobacter pylori. The common symptoms of patients with chronic gastritis and peptic ulcer are: after eating, the upper abdomen is full, uncomfortable or painful, often accompanied by other adverse symptoms such as belching, abdominal distension, acid regurgitation, anorexia, etc. Some patients also have recurrent severe abdominal pain, and small amount of upper gastrointestinal hemorrhage. Helicobacter pylori disease is acquired. The mode of transmission is not well defined, but the most likely routes are oral-oral, fecal-oral, pet-human transmission, as can be demonstrated by the following experiments: 1. detecting DNA of helicobacter pylori from saliva, plaque and feces by using PCR; 2. separating helicobacter pylori from plaque and feces; 3. the same helicobacter pylori strains were isolated from pet excreta from the same residence.
There are many methods for detecting helicobacter pylori infection, such as biopsy, isolated culture of helicobacter pylori, rapid urease test, urea breath test, urinary ammonia excretion test, serology test, polymerase chain reaction, etc. Therefore, on the basis of the above, it is necessary to develop a rapid and simple detection method.
Disclosure of Invention
In order to make up the defects of the prior art, the invention provides a monoclonal antibody of helicobacter pylori and a preparation method thereof; the second purpose of the invention is to provide a helicobacter pylori detection kit prepared by using the monoclonal antibody prepared by the invention.
On one hand, the invention discloses a helicobacter pylori UreB epitope polypeptide, and the amino acid sequence of the epitope polypeptide is GEVITRTWQTADKNKKEFGRLKEEKG.
On the other hand, the invention also discloses a monoclonal antibody of the epitope polypeptide, wherein the nucleotide sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown as SEQ ID NO.1 and SEQ ID NO. 2.
In still another aspect, the present invention also discloses a kit for detecting helicobacter pylori antibodies, which comprises an effective amount of the epitope polypeptide of claim 1, the monoclonal antibody of claim 2 and a matched detection reagent.
Preferably, the epitope polypeptide in the kit of the present invention is conjugated to BSA before use, and the coating amount of the epitope polypeptide conjugated to BSA is 5. mu.g/ml.
Preferably, the monoclonal antibody of the invention is labeled with HRP before use, and the HRP-labeled monoclonal antibody is diluted 20000 times at the time of use.
On the other hand, the invention also discloses application of the epitope polypeptide in preparing a helicobacter pylori diagnostic reagent.
On the other hand, the invention also discloses the application of the monoclonal antibody in preparing a helicobacter pylori diagnostic reagent.
On the other hand, the invention also discloses application of the antigen epitope polypeptide in preparation of helicobacter pylori antigen epitope vaccines.
The epitope polypeptide prepared by the invention has good immunogenicity and specificity; the monoclonal antibody prepared by the invention has good specificity and sensitivity; the diagnostic kit prepared by the epitope polypeptide and the monoclonal antibody has good specificity, sensitivity and accuracy, and is suitable for large-range application
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FIG. 1 is a SDS-PAGE image of the monoclonal antibody, and 1 is the purified monoclonal antibody.
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Example 1: design and screening of helicobacter pylori urease B subunit (UreB) epitope polypeptide
UreB protein of UniProtKB-P69996(URE1_ HELPY) is selected, and 9 epitope polypeptides (Table 1) are designed and synthesized through analysis and research of the protein sequence. The 9 antigen epitope polypeptides (because the antigen epitope polypeptides are haptens, the antigen epitope polypeptides are coupled with BSA (using a conventional glutaraldehyde method) before immunization) to immunize mice respectively, and the serum of the mice is collected to carry out ELISA antibody titer detection, so as to screen the antigen epitope polypeptides with the highest antibody titer. The epitope polypeptide 9 was tested to have the highest antibody titer (table 2), and therefore, epitope polypeptide 9 was selected as the focus of the subsequent studies.
And (3) ELISA antibody titer detection: preparing a UreB protein (described in CN 112794902A example 1) coated ELISA plate by using a conventional prokaryotic expression foreign protein method, wherein the enzyme is coated at 100 ng/hole and 0.1 ml/hole and is coated overnight at 4 ℃; after washing for 3 times, sealing with 5% skimmed milk, and incubating at 37 deg.C for 2 h; mouse serum was serially diluted 10-fold (10-10) with PBS7) Then added into a coated enzyme label plate, 0.1 ml/hole,incubating at 37 ℃ for 1 h; washing for 3 times, adding HRP-labeled goat anti-mouse IgG secondary antibody diluted by 1:5000 times, 0.1 ml/hole, and incubating at 37 deg.C for 30 min; washing for 3 times, adding 0.1ml TMB single component color developing solution (purchased from Beijing Soilebao Biotech Co., Ltd.), and incubating at 37 deg.C for 10 min; adding stop solution (2M sulfuric acid), and measuring the OD450nm value; a positive result was determined when the OD450nm value was greater than 0.1.
TABLE 1 UreB epitope polypeptides
Serial number Sequence Position ELISA antibody titer
1 KEYVSMYGPTTGDKVRLGDTDLIAEV 7-32 1:102
2 GDKVRLGDTDLIAEVEHDYTIYGEEL 18-43 1:103
3 IYGEELKFGGGKTLREGMSQSNNPSK 38-63 1:102
4 IYKADIGIKDGKIAGIGKGGNKDMQD 81-106 1:102
5 TITPGRRNLKWMLRAAEEYSMNLGFL 171-196 1:104
6 IGFKIHEDWGTTPSAINHALDVADKY 216-241 1:103
7 AIAGRTMHTFHTEGAGGGHAPDIIKV 264-289 1:10
8 MLMVCHHLDKSIKEDVQFADSRIRPQ 317-342 1:103
9 GEVITRTWQTADKNKKEFGRLKEEKG 370-395 1:105
Example 2: preparation and detection of helicobacter pylori resistant urease B subunit (UreB) epitope polypeptide monoclonal antibody
Screening hybridoma cells: immunizing a BalB/C mouse with the synthesized epitope polypeptide 9, separating the splenocytes of the mouse after three times of immunization, fusing the splenocytes with myeloma cells Sp2/0, cloning and screening by a limiting dilution method, screening cell strains with high antibody titer and good shapes, continuously subcloning by the limiting dilution method until monoclonal cells are obtained, and carrying out expanded culture on the monoclonal cells and storing the monoclonal cells in liquid nitrogen. Through multiple rounds of detection and screening, 1 better positive hybridoma cell is finally obtained and compiled into hybridoma cell 1A 2.
Preparing ascites: taking 6-8 weeks old BALB/c female mice, and injecting 0.5ml per mouse by intraperitoneal injection, and after 7 days, injecting 1 × 10 mice by intraperitoneal injection6And (3) after 7-10 days of hybridoma cells 1A2, aseptically extracting ascites of the mouse by using an injector, centrifuging at 12000rpm at 4 ℃ for 20min, collecting supernatant, namely ascites antibody, subpackaging the harvested supernatant, and storing at 2 ml/tube below-70 ℃ for later use.
Antibody purification: the prepared ascites antibody is purified by a conventional caprylic acid-ammonium sulfate precipitation method (see Zhouyu jade and the like, research on a purification method of mouse ascites IgG monoclonal antibody, Heilongjiang animal veterinarian, No. 10 in 2006). 20 mu l of the purified ascites antibody is taken, and purity test is carried out by using SDS-PAGE, and the result shows that the purity of the SDS-PAGE of the monoclonal antibody is more than 95 percent (figure 1); and detecting the protein concentration of the purified antibody by using a BCA kit, wherein the protein concentration is 2.12 mg/ml.
Sequencing of monoclonal antibody: see the Chinese patent (CN 111393525B) for analysis and determination of heavy chain variable region and light chain variable region of the prepared monoclonal antibody. Through detection and analysis, the heavy chain variable region sequence of the monoclonal antibody is shown as SEQ ID NO.1, and the light chain variable region sequence is shown as SEQ ID NO. 2. The heavy chain variable region sequence and the light chain variable region sequence were analyzed, and the results are shown in Table 2.
TABLE 2 sequence information of monoclonal antibody variable regions
Figure BDA0003559974870000041
Example 3: detection of helicobacter pylori antibodies
Preparing an enzyme label plate: the screened epitope polypeptide is adopted to coat the ELISA plate, because the epitope polypeptide is hapten, the epitope polypeptide is coupled with BSA (by using a conventional glutaraldehyde method) to obtain antigen before coating, the antigen is coated on the ELISA plate at a dose of 5 mu g/ml (0.1 ml/hole), and the ELISA plate is coated overnight at 4 ℃; after washing, 0.2ml of blocking solution (PBST solution containing 2% BSA) was added per well, blocked overnight at 4 ℃, washed, blotted dry, and stored at-20 ℃ for future use.
Enzyme-labeled antibody preparation: the monoclonal antibodies were HRP labeled using a modified sodium periodate method as follows: placing 0.1ml HRP and 0.05ml NaIO4(0.06M) in a reaction tube, and reacting for 20min at room temperature in a dark place; then adding 0.05ml of glycol solution, and reacting for 20min at room temperature in a dark place; then dialyzed overnight at 4 ℃ against 1L of 0.01M carbonate buffer pH 9.5; adding 0.05mg of monoclonal antibody, and stirring gently at room temperature in a dark place for 2 hours; then 0.025ml of NaBH4 solution (4mg/ml) is added to react for 2h at 4 ℃; dialyzing with 1L PBS overnight at 4 deg.C; centrifuging at 4000rpm and 4 ℃ for 30min, and taking the supernatant to obtain the monoclonal antibody marked by the HRP. And adding glycerol with the same volume as the labeled monoclonal antibody, and storing at-20 ℃ for later use. Before use, 20000 times diluted by a sealing liquid and stored at 4 ℃ for later use.
Detection by the kit: 1) diluting a serum sample 1:1, adding the diluted serum sample into an enzyme label plate at a concentration of 0.1 ml/hole, adding 0.1ml of positive control (prepared monoclonal antibody with the concentration of 1 mu g/ml) and 0.1ml of negative control (confining liquid) respectively, and incubating for 60 minutes at 37 ℃; 2) washing, adding an enzyme-labeled antibody with the concentration of 0.1 ml/hole, and incubating for 30 minutes at 37 ℃; 3) adding developing solution (TMB developing solution from Biotech, Inc. of Beijing Solaibao) 0.1 ml/well after washing, and incubating at 37 deg.C for 10 min; 4) adding stop solution (2M H)2SO4) 0.1 ml/hole, mixing uniformly, immediately placing the coated plate in an enzyme-labeling instrument, and reading the OD450nm value under the wavelength of 450 nm; calculating S/N values from OD450nm values (sample OD450nm value/negative control OD450nm value); 5) when the OD450nm reading of each well of the negative control well is greater than 1.0, the maximum difference between the wells should be<0.3, Positive control wells should read OD450nm per well<0.3, the test is established; 6) when the S/N value is greater than 0.5, the sample is judged to be negative, and when the S/N value is less than or equal to 0.5, the sample is judged to be positive.
The application of the kit comprises: the inventors collected 30 parts of serum from a helicobacter pylori-infected patient at a certain hospital in Guangdong and 45 parts of normal serum. The prepared kit is used for detecting 75 parts of serum respectively, and the result shows that 30 parts of serum of a helicobacter pylori infected patient detected by the kit is positive, and 45 parts of normal serum is negative, so that the kit has good accuracy, specificity and sensitivity and is suitable for large-scale application.
In addition, the helicobacter pylori UreB epitope polypeptide can generate specific reaction on serum of a patient infected by helicobacter as an antigen, and the antigen epitope polypeptide has good immunogenicity aiming at UreB and can be used as one of candidate polypeptides of an epitope vaccine.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Stadium Kouyn
<120> helicobacter pylori monoclonal antibody and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 420
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aagggtgttc agagcgagga taagctggaa gaatctggcg gaggtctggt tcagccaggc 60
ggtagtatga aactgagctg cgttgccagc ggctttacaa ttgagaacta ctggatgaat 120
tgggtatcac agtctcctga gaagggcctt gattggagcg cagagcggag gctgaaaagt 180
aacaactatg tgacacacta cgcatgtagc gttaagggtc gctttggcat cagcagggac 240
gactccaaaa atagtgtgta cctgcagatg aataaccccc ggcctgagga caccggtatc 300
tattactgca cccccatcta cagtcctttt gcttattggg ggggtgggac cctcgtttca 360
gtttctgccg ccaagactac accaccttca gtgtatccaa acgctcccgg atcagctgcc 420
<210> 2
<211> 348
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gacggggaca ttgtgatgac acagtcccag aaaatgtcca ccagtgtggg attccgcgta 60
tctgtgacct gtaaggcttc acagaatgtt aacaatgatg tggcttggca ccagcagaaa 120
cctggccaga gccctaaggc cctgttctac tccgcttcct gtagatatag cggagtccca 180
gaccgcttta ccggatctgg ctacggcaca gacttcaccc tgacttcctc taacgtgcaa 240
atgaaggatc tcgcagagta tttttgccag cagtataact cattcccata tactgaaggc 300
gggggcacca agctcgagat caaacgagcc agggcagctc cgactgtg 348

Claims (8)

1. The helicobacter pylori UreB epitope polypeptide is characterized in that the amino acid sequence of the epitope polypeptide is GEVITRTWQTADKNKKEFGRLKEEKG.
2. A monoclonal antibody against the epitope polypeptide of claim 1, wherein the variable regions of the heavy chain and the light chain of the monoclonal antibody have the nucleotide sequences shown in SEQ ID No.1 and SEQ ID No. 2.
3. A kit for detecting helicobacter pylori antibodies, which is characterized in that the kit comprises effective amounts of the epitope polypeptide of claim 1, the monoclonal antibody of claim 2 and matched detection reagents.
4. The kit of claim 3, wherein the epitope polypeptide is conjugated to BSA before use, and the amount of the epitope polypeptide conjugated to BSA is 5. mu.g/ml.
5. The kit of claim 3, wherein the monoclonal antibody is labeled with HRP prior to use, and wherein the HRP-labeled monoclonal antibody is diluted 20000-fold at the time of use.
6. Use of the epitope polypeptide of claim 1 in the preparation of a helicobacter pylori diagnostic reagent.
7. Use of the monoclonal antibody of claim 2 for the preparation of a diagnostic reagent for helicobacter pylori.
8. The use of the epitope polypeptide of claim 1 in the preparation of a helicobacter pylori epitope vaccine.
CN202210286040.1A 2022-03-23 2022-03-23 Helicobacter pylori monoclonal antibody and application thereof Withdrawn CN114480356A (en)

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Cited By (6)

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CN115267208A (en) * 2022-09-27 2022-11-01 上海芯超生物科技有限公司 Antigen and kit for detecting helicobacter pylori antibody and preparation method thereof
CN117050169A (en) * 2023-08-14 2023-11-14 苏州东抗生物科技有限公司 Monoclonal antibody for UreA protein and application thereof
CN117164707A (en) * 2022-08-05 2023-12-05 陕西茗奥幕生物科技有限公司 Preparation method of probiotics and application of probiotics in treating gastrointestinal diseases
EP4338749A1 (en) * 2022-09-14 2024-03-20 Iguana Biotechnology GmbH Vaccine against helicobacter pylori infection
WO2024066594A1 (en) * 2022-09-28 2024-04-04 北京金沃夫生物工程科技有限公司 Test paper and kit for detecting helicobacter pylori
GB2627550A (en) * 2022-09-28 2024-08-28 Beijing Jinwofu Bioengineering Tech Co Ltd Test strip and kit for detecting helicobacter pylori(HP)

Cited By (8)

* Cited by examiner, † Cited by third party
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CN117164707A (en) * 2022-08-05 2023-12-05 陕西茗奥幕生物科技有限公司 Preparation method of probiotics and application of probiotics in treating gastrointestinal diseases
EP4338749A1 (en) * 2022-09-14 2024-03-20 Iguana Biotechnology GmbH Vaccine against helicobacter pylori infection
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