CN117362424A - Helicobacter pylori resistant monoclonal antibody and application thereof - Google Patents
Helicobacter pylori resistant monoclonal antibody and application thereof Download PDFInfo
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- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/121—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Helicobacter (Campylobacter) (G)
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56922—Campylobacter
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G01N2333/205—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Campylobacter (G)
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Abstract
The invention provides an anti-helicobacter pylori monoclonal antibody and application thereof, wherein the anti-helicobacter pylori monoclonal antibody comprises a heavy chain variable region shown in SEQ ID NO.7 and a light chain variable region shown in SEQ ID NO. 8. The monoclonal antibody is a murine monoclonal antibody, the monoclonal antibody has good specificity and high affinity, and the anti-helicobacter pylori monoclonal antibody has important application value in preparing helicobacter pylori detection products.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an anti-helicobacter pylori monoclonal antibody and application thereof.
Background
Helicobacter pyloriHelicobacter Pylori，H. Pylori) Is a gram negative pathogen with strong infectivity and wide transmission range, often causes intestinal and stomach diseases of human body, and even causes chronic gastritis, peptic ulcer, lymphoma, liver cancer, gastric cancer and other diseases. Helicobacter pylori has been listed as a first class of carcinogen by the world health organization international cancer research institute as early as 2017. In recent years, helicobacter pylori has exploded worldwide, the number of infected people has increased significantly, and the global infection has reached about 50%. Helicobacter pylori infection has a large number of people, is easy to infect and is difficult to radically cure, and has become a great difficulty. Therefore, early discovery, early treatment, early control are particularly important, and research and development of a novel diagnosis method for helicobacter pylori infection is urgent.
Monoclonal antibodies are highly homogeneous antibodies raised from a single B cell clone, directed against only one specific epitope, and are referred to as monoclonal antibodies. The hybridoma (hybridoma) antibody technology is generally prepared by fusing sensitized B cells with the capability of secreting specific antibodies and myeloma cells with unlimited reproductive capability into B cell hybridomas on the basis of a cell fusion technology. By culturing a cell population with a single hybridoma cell having such characteristics, a monoclonal antibody, which is a specific antibody against an epitope, can be produced. The murine monoclonal antibody has strong affinity and specificity, and is the most widely used monoclonal antibody at present.
The immunodetection technique is a method for realizing qualitative or quantitative detection of antigen or antibody by detecting a marker labeled on a reactant by utilizing specific binding reaction between antigen and antibody. According to the labeling substance, it is classified into Enzyme-linked immunosorbent assay (ELISA), immunofluorescence detection, chemiluminescent immunoassay, immunomicrosphere and immunocolloidal gold. The ELISA technology has the characteristics of simple operation, high sensitivity and short detection time, and is widely used in clinical detection and scientific research.
The antibody with excellent performance is the basis of the immunodetection technology, and only the antibody with good performance can develop the immunodetection kit with excellent performance. Therefore, the provision of a monoclonal antibody against helicobacter pylori with high affinity has great significance for helicobacter pylori detection.
Disclosure of Invention
Aiming at the defects existing in the prior art, the invention aims to provide an anti-helicobacter pylori monoclonal antibody and application thereof, wherein the monoclonal antibody is a murine monoclonal antibody, and the anti-helicobacter pylori monoclonal antibody has important application value in preparing helicobacter pylori detection products.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides an anti-helicobacter pylori monoclonal antibody comprising a heavy chain variable region and a light chain variable region;
the heavy chain variable region comprises a heavy chain CDR1 shown in SEQ ID NO.1, a heavy chain CDR2 shown in SEQ ID NO.2 and a heavy chain CDR3 shown in SEQ ID NO. 3;
the light chain variable region comprises a light chain CDR1 shown in SEQ ID NO.4, a light chain CDR2 shown in SEQ ID NO.5 and a light chain CDR3 shown in SEQ ID NO. 6.
SEQ ID NO.1：SDYWN。
SEQ ID NO.2：YISYRGTTYYNPSLKS。
SEQ ID NO.3：GANSFDY。
SEQ ID NO.4：GAAENIYGALN。
SEQ ID NO.5：GATNLAD。
SEQ ID NO.6：QNVLTNPFT。
The monoclonal antibody has good specificity and high affinity; the monoclonal antibody is a murine monoclonal antibody; the monoclonal antibody does not generate cross reaction with other enteropathogenic bacteria (including general bacillus proteus, klebsiella pneumoniae, enteropathogenic escherichia coli, salmonella enteritidis, salmonella typhimurium, staphylococcus aureus, escherichia coli and BSA), and has strong specificity; the monoclonal antibody has strong binding capacity to helicobacter pylori antigen, high affinity, high potency to helicobacter pylori antigen and EC50 value up to 0.003976 ng/. Mu.L.
Preferably, the amino acid sequence of the heavy chain variable region comprises the sequence shown in SEQ ID NO. 7.
Preferably, the amino acid sequence of the light chain variable region comprises the sequence shown in SEQ ID NO. 8.
SEQ ID NO.7：
EVQLQESGPGLAKPSQTLSLTCSVTGYSITSDYWNWIRIFPGNKLEHMGYISYRGTTYYNPSLKSRISITRDTSKNQFYLQLNSVTTEDTATYFCAGGANSFDYWGQGTTLTVSS
SEQ ID NO.8：
DIQMTQSPASLSASVGETVTITCGAAENIYGALNWYQRKQEKSPQLLIYGATNLADGMSSRFSGSGSGRQYSLKISSLHPDDVATYYCQNVLTNPFTFGGGTKLEIK
Preferably, the anti-helicobacter pylori monoclonal antibody further comprises any one or a combination of at least two of murine IgG1, igG2, igG3, or IgG4 constant regions, preferably murine IgG1 constant regions.
According to the invention, female BALB/c female mice with 6-8 weeks of age are selected as experimental animals, and monoclonal antibodies are prepared; according to the invention, helicobacter pylori is used as an antigen for immunization, and the obtained spleen cells are subjected to cell fusion with myeloma cells to obtain the murine monoclonal antibody, so that the stability is good, and the monoclonal antibody has strong affinity to the helicobacter pylori; the monoclonal antibody prepared by the invention has good specificity and strong affinity with helicobacter pylori, but does not generate cross reaction with other pathogenic bacteria such as common proteus, klebsiella pneumoniae, enteropathogenic escherichia coli, salmonella enteritidis, salmonella typhimurium, staphylococcus aureus, escherichia coli and the like, and can be potentially applied to antigen detection of helicobacter pylori, detection and identification of helicobacter pylori infection clinical samples and the like.
In a second aspect, the invention provides a nucleic acid molecule encoding the anti-helicobacter pylori monoclonal antibody of the first aspect.
Preferably, the nucleotide sequence encoding the heavy chain variable region of the anti-helicobacter pylori monoclonal antibody comprises the sequence shown in SEQ ID NO. 9; the nucleotide sequence of the light chain variable region of the anti-helicobacter pylori monoclonal antibody comprises a sequence shown in SEQ ID NO. 10.
SEQ ID NO.9：
GAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGCCAAGCCTTCTCAGACCCTGAGCCTGACCTGTTCTGTGACCGGCTACAGCATCACCAGCGACTACTGGAACTGGATCAGGATCTTCCCCGGCAACAAGCTGGAGCACATGGGCTACATCAGCTACAGGGGCACCACCTACTACAACCCCAGCCTGAAGAGTCGGATCTCCATCACTCGGGACACATCCAAGAACCAGTTCTACCTGCAGCTGAACTCTGTGACCACCGAGGACACCGCCACCTACTTCTGCGCCGGCGGCGCCAACAGCTTCGACTACTGGGGCCAGGGCACCACTCTCACTGTCTCCTCA。
SEQ ID NO.10：
GACATCCAGATGACTCAGTCTCCAGCTTCACTGTCTGCATCTGTGGGCGAGACCGTGACCATCACCTGCGGCGCCGCCGAGAACATCTACGGCGCCCTGAACTGGTACCAGCGGAAACAGGAGAAGAGCCCTCAGCTCCTGATCTATGGTGCCACCAACCTGGCCGACGGCATGTCATCGAGGTTCAGTGGCAGTGGATCTGGTAGACAGTATAGCCTGAAGATCAGCAGCCTGCACCCCGACGACGTTGCAACGTATTACTGTCAAAATGTGCTGACCAACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAA。
In a third aspect, the present invention provides an expression vector comprising a nucleic acid molecule according to the second aspect.
In a fourth aspect, the invention provides a host cell comprising at least one copy of the expression vector of the third aspect, or having integrated into its genome the nucleic acid molecule of the second aspect.
Preferably, the host cell comprises a 293T cell or CHO cell.
In a fifth aspect, the invention provides a pharmaceutical composition comprising an anti-helicobacter pylori monoclonal antibody according to the first aspect.
Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
In a sixth aspect, the present invention provides a kit for detecting helicobacter pylori, the kit for detecting helicobacter pylori comprising the anti-helicobacter pylori monoclonal antibody of the first aspect.
Preferably, the kit for detecting helicobacter pylori further comprises any one or a combination of at least two of a positive control, a negative control, an antibody diluent, a chromogenic solution, a stop solution, a blocking solution and a washing solution.
In a seventh aspect, the present invention provides the use of any one or a combination of at least two of the anti-helicobacter pylori monoclonal antibody of the first aspect, the nucleic acid molecule of the second aspect, the expression vector of the third aspect, the host cell of the fourth aspect or the pharmaceutical composition of the fifth aspect for the preparation of a medicament for the treatment of helicobacter pylori infection disease and/or an assay product.
Compared with the prior art, the invention has the following beneficial effects:
the monoclonal antibody has good specificity and high affinity; the monoclonal antibody is a murine monoclonal antibody; the monoclonal antibody does not generate cross reaction with other pathogenic bacteria (including general Proteus, klebsiella pneumoniae, enteropathogenic escherichia coli, salmonella enteritidis, salmonella typhimurium, staphylococcus aureus, escherichia coli and BSA), and has strong specificity; the monoclonal antibody has strong binding capacity to helicobacter pylori antigen, high affinity, high potency to helicobacter pylori antigen and EC50 value up to 0.003976 ng/. Mu.L.
Drawings
FIG. 1 is a diagram showing the result of SDS-PAGE of the monoclonal antibodies in example 6.
FIG. 2 shows the results of the detection of the affinity activity of the monoclonal antibody of example 7.
FIG. 3 shows the results of the cross-reaction in example 8.
Detailed Description
The technical scheme of the invention is further described by the following specific embodiments. It will be apparent to those skilled in the art that the examples are merely to aid in understanding the invention and are not to be construed as a specific limitation thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1 antigen preparation
The preparation method of helicobacter pylori antigen is as follows:
(1) Recombinant plasmid construction
According to the NCBI published coding gene sequence, strep tag sequence is added, and based on pcDNA3.4 map, eukaryotic expression recombinant plasmid pcDNA3.4-HP is constructed by gene synthesis of Anshengda biotechnology Co.
(2) Endotoxinfree plasmid large extract
Plasmid DNA was extracted according to the protocol using endotoxin-free plasmid extraction kit (available from Tiangen Biochemical technologies Co., ltd., accession number DP117#), and cell transfection was performed after qualification of concentration measurement and purity verification.
（3）Expi293 TM The cell transfection procedure is as follows:
(a) Cell density and cell viability were determined using a fully automatic cytometer and trypan blue staining. Cell viability > 95% and viable cell density 3.0X10 6 cells/mL were transfected and therefore diluted to 3 x 10 with freshly pre-warmed Expi293 ™ expression medium 6 Live cells/mL, then shake the flask gently to mix the cells.
(b) Before use, the Expifectamine293 reagent bottle was gently inverted 4-5 times to ensure adequate mixing.
(c) The plasmid DNA was diluted with Opti-Plex ™ complex buffer (or Opti-MEM ™ I medium) and then gently shaken or mixed upside down. The total plasmid DNA concentration in the transfected culture was 1.0. Mu.g/mL.
(d) The Expi Fectamine ™ 293 reagent was diluted with Opti-Plex ™ multiplex buffer (or Opti-MEM ™ I medium) and then gently shaken or mixed upside down. Incubate at room temperature for 5 min.
(e) Diluted Expi Fectamine ™ 293 reagent was added to the diluted plasmid DNA and then mixed gently with shaking or upside down.
(f) The epi Fectamine ™ 293/plasmid DNA complex was incubated at room temperature for 10-20 min.
(g) Slowly transferring the complex into cells, shaking culture flask during addition, and placing on shaking table at a relative humidity of 80% or more and CO of 8% 2 Cells were cultured in a 37℃shaker.
(h) After transfection, 18-22 h, the transfection enhancer 1 of the Expi Fectamine ™ 293 and the transfection enhancer 2 of the Expi Fectamine ™ 293 are added into the transfection flask, and the flask is gently shaken during the addition. Immediately placing the flask back on the shaking table at a relative humidity of 80% or more and 8% CO 2 Cells were cultured in a 37℃shaker.
(4) The antigen protein is purified by affinity chromatography, STarm Streptactin Beads FF column materials are selected for purification, and the specific experimental operation of purification is as follows:
(a) Filling a gravity column: and (3) taking a gravity chromatographic column with proper specification, loading the column into a lower gasket, adding a proper amount of pure water to rinse the column tube and the gasket, and closing a lower outlet. STarm Streptactin Beads 4FF is mixed evenly, a proper amount of slurry is sucked by a gun head and added into a gravity column, and a lower outlet is opened to drain protective liquid. Adding proper amount of pure water to wash medium, and closing the lower outlet after the liquid in the column tube is drained by gravity. And (3) loading the rinsed upper gasket, ensuring that no gap exists between the gasket and the filler, and keeping the gasket horizontal. The filled gravity column can be directly added with balance liquid for balancing.
(b) Sample purification: the packed STarm Streptactin Beads 4FF gravity column is balanced by 5 times of column volume balancing liquid, so that the packing is in the same buffer system as the target protein, and the process is repeated for 2 to 3 times. And (3) adding the sample into a balanced gravity column, wherein the retention time of the sample is at least 2 min, so that the sample is ensured to be fully contacted with a medium, and effluent liquid is collected, so that the sample can be repeatedly loaded to increase the binding efficiency. Eluting with 10-15 times of column volume of eluting solution (0.01 moL/L PBS solution), removing nonspecifically adsorbed impurity protein, and collecting eluting solution. Eluting with 5-10 times of column volume eluent (0.1 moL/L glycine-HCl solution with pH of 3.0), collecting in sections, collecting one tube for each column volume, neutralizing with Tris-HCl solution (pH of 8.5), and detecting respectively, which can ensure that all the conjugated target proteins are eluted, and can obtain high-purity and high-concentration proteins.
(c) Ultrafiltration: sucking the purified protein into a 50 mL ultrafiltration tube (ultrafiltration tube diameter 30 kD), placing into a centrifuge according to the use requirement of the ultrafiltration tube, and centrifuging at 6000 r/min for 20 min after balancing. After centrifugation, the lower filtrate was discarded, the upper liquid was pipetted into a clean 50 mL centrifuge tube, and 0.01 moL/L PBS was added thereto. Repeating the steps for 5-10 times to obtain the antigen protein with proper concentration. The obtained helicobacter pylori antigen protein can be subjected to the next experiment after the concentration measurement and the purity verification are qualified.
EXAMPLE 2 animal immunization
(1) The mice immunization protocol was as follows:
(a) The purified protein with good titer and high purity is used as an antigen to immunize a mouse, when the antigen protein is injected into the abdominal cavity of the mouse for the first time, the protein is mixed with Freund's complete adjuvant in equal proportion, the mixture is fully emulsified and then injected, and the immune dose of each mouse is 50 mug protein.
(b) After two weeks, the second immunization was performed by mixing the protein with Freund's incomplete adjuvant in equal proportions, emulsifying it sufficiently, and injecting it through the abdominal cavity, with an immunization dose of 50. Mu.g protein per mouse.
(c) After two more weeks, the procedure of (b) above was repeated.
(d) After one week of interval, antisera were collected by mouse orbital vein bleeding and serum titers were determined by indirect ELISA.
(e) After one week of interval, the above step (b) is repeated again.
(f) The non-emulsified proteins were directly injected into the abdominal cavity of mice with matched serum titers after screening, so that the mice were boosted and cell fusion was performed three days later.
The purified helicobacter pylori antigen is adopted as an artificial immunogen, mice are immunized according to the immunization scheme, and antisera are collected through blood collection of the orbit veins of the mice. Mice are put intoSerum sample is adjusted to initial concentration of 10 ng/. Mu.L, and is diluted in three times for gradient dilution, and Log is taken 10 Values are plotted.
Serum titers were then determined by indirect ELISA. And the protein concentration is simply measured by using an ultraviolet protein quantitative instrument, and the optimal antiserum is simply brushed according to the titer and the protein concentration value.
(2) The method for determining the potency of the antiserum is as follows:
(a) Helicobacter pylori antigen is taken as coating antigen, added into a 96-well plate, and coated overnight at 37 ℃ with the coating concentration of 100 ng/well. After coating, the liquid in the wells was discarded, 200. Mu.L of PBST was added to each well, the plates were washed three times for 5 min each, and the wells were dried by pipetting.
(b) Adding a blocking solution prepared by 3% BSA, incubating at 37 ℃ for 2 h, throwing away the liquid, discarding the blocking solution after blocking, adding 200 mu L of PBST into each hole, washing the plate for three times, each time for 5 min, and beating to dry for later use.
(c) The prepared antibody serum was subjected to gradient dilution, added to an ELISA plate, added with 100. Mu.L per well, incubated at 37℃for 1 h, and then washed three times with PBST, and allowed to stand for 3 min each time.
(d) HRP-labeled goat anti-mouse IgG was used as an enzyme-labeled antibody, diluted 1:5000, added 100. Mu.L per well, incubated 1 h at 37℃and then washed three times with PBST, with 40 s standing each time.
(e) 100. Mu.L TMB was added to each well and allowed to stand, and incubated at 37℃for 15 min.
(f) The reaction was terminated by adding 50. Mu.L of stop solution to each well, and OD was measured by an ELISA reader 450 Values.
EXAMPLE 3 preparation of murine monoclonal antibody
And (3) performing titer detection on the prepared mouse antiserum, and under the condition of qualified titer, respectively performing cell fusion by using qualified mouse spleen to prepare monoclonal hybridoma cell strains, wherein the specific preparation method is as follows:
(1) Preparation of immune spleen cells
(a) And (3) performing eyeball blood collection on a mouse with serum titer meeting the requirement, collecting serum, then performing dislocation sacrifice on cervical vertebra, soaking in 75% (v/v) ethanol for 10 min, and performing disinfection treatment.
(b) The mice were transferred to an ultra clean bench, the abdomen of the mice was cut with surgical scissors, the tissues around the spleen were removed, the spleen was completely exposed, and the spleen was removed with forceps.
(c) The spleen was placed in a cell culture solution, adipose tissue on the spleen was removed, and red blood cells were gently washed away.
(d) The spleen was placed on a cell sieve, gently ground with forceps, continuously rinsed with cell culture fluid, and blown uniformly. 1000 Centrifuging for 10 min at r/min, and discarding supernatant.
(e) Re-adding cell culture liquid to re-suspend the cells to make cell number reach 10 8 Left and right, for standby.
(2) Cell fusion
(a) Taking SP2/0 myeloma cells in logarithmic phase, mixing with the obtained spleen cells, washing with cell culture solution without fetal bovine serum once, centrifuging at 1000 r/min for 10 min, and discarding supernatant.
(b) Adding polyethylene glycol solution, maintaining at 37deg.C and 90-s, stopping reaction with cell culture solution without fetal calf serum, centrifuging at 1000 r/min for 10 min, and discarding supernatant.
(c) Resuspension with HAT selective culture medium containing 20% fetal bovine serum, adding 100 μl of cells per well into 96-well plate, culturing in cell culture box at 37deg.C under CO 2 The content was 5.0%.
(3) Cell monoclonalization and screening
Diluting cells with good growth state in 96-well plate with cell culture solution to 1-3 cells/mL, adding into 96-well plate, culturing in cell culture box at 37deg.C, and CO 2 The content was 5.0%. And (5) numbering each cell strain respectively, and selecting the cell strain positive in the supernatant of the culture solution for expansion culture. Finally obtaining the hybridoma cell strain.
(4) Screening for hybridoma cells
The obtained hybridoma cells are screened by ELISA method, and a maximum of 10 polyclonal antibody cell strains aiming at helicobacter pylori antigens are found, and the cell strains can generate specific monoclonal antibodies with high affinity aiming at the respective antigens.
After the cell fusion step, two parental cells and three randomly fused cells are present in the medium, and in order to obtain a hybridoma cell line capable of secreting the antibody of interest, the successfully fused hybridoma cells must be isolated from the multitude of cells. B lymphocytes cannot survive in vitro for a long period of time, and only myeloma cells and fusion cells thereof need to be removed, so that the fused cells need to be cultured by HAT medium, and hybridoma cells are selectively retained.
The growth condition of cells can be observed on the 5 th day after fusion, and the cell culture supernatant can be detected by adopting an indirect ELISA method and positive hybridoma cell strains can be screened for cloning culture on the 10 th to 14 th days. And (3) cloning and culturing the positive hybridoma cells by adopting a limiting dilution method. And amplifying the positive hybridoma cells with the strongest titer of the detection result to a strain when the cell positive rate reaches 100%. Titers of culture supernatants of the hybridoma cell lines of the fixed strains were measured by ELISA, and the expanded monoclonal hybridoma cell lines were frozen in liquid nitrogen.
Performing specificity and affinity tests on the hybridoma cell supernatants, and selecting hybridoma cells from which to pair; carrying out recombinant expression on the selected four-hole polyclonal hybridoma cell strain to obtain a monoclonal antibody; and (3) carrying out sandwich pairing on the obtained purified antibodies, establishing a sandwich detection system of helicobacter pylori antigens, and finally obtaining the monoclonal antibodies with the best pairing respectively.
EXAMPLE 4 murine monoclonal antibody sequencing
(1) Isolation of Total RNA from hybridoma cells
After homogenization of the hybridoma cells, TRIzol is added and the homogenate can be separated into a clear upper aqueous layer (containing RNA), a phase interface and a red lower organic layer (containing DNA and protein). RNA was then precipitated from the aqueous layer with isopropanol. The DNA was precipitated from the organic layer with ethanol. Proteins were precipitated from phenol-ethanol supernatant using isopropanol precipitation. Washing the precipitated RNA, removing impurities, and suspending again for later use.
(2) Reverse transcription of Total RNA into cDNA
dNTPs are used as substrates, RNA is used as a template, tRNA is used as a primer, and a cDNA single strand complementary to the RNA template is synthesized on the 3' -end of the tRNA in the 5 '. Fwdarw.3 ' direction, and forms an RNA-cDNA hybrid with the RNA template. Then, RNA strand is hydrolyzed under the action of reverse transcriptase, and cDNA is used as template to synthesize the second DNA strand. To this end, total RNA was reverse transcribed into cDNA.
(3) Rapid Amplification of CDNA Ends (RACE)
Antibody fragments of the heavy and light chains were amplified according to standard procedures for Rapid Amplification of CDNA Ends (RACE). The amplified antibody fragments were cloned separately into standard cloning vectors. Colony PCR was performed to screen clones with inserts of the correct size. The antibody sequence is obtained. The nucleotide sequence of the antibody is shown below:
the nucleotide sequence of the heavy chain variable region of the anti-helicobacter pylori monoclonal antibody comprises a sequence shown in SEQ ID NO. 9; the nucleotide sequence of the light chain variable region of the anti-helicobacter pylori monoclonal antibody comprises a sequence shown in SEQ ID NO. 10.
SEQ ID NO.9：
GAGGTGCAGCTGCAGGAGAGCGGCCCCGGCCTGGCCAAGCCTTCTCAGACCCTGAGCCTGACCTGTTCTGTGACCGGCTACAGCATCACCAGCGACTACTGGAACTGGATCAGGATCTTCCCCGGCAACAAGCTGGAGCACATGGGCTACATCAGCTACAGGGGCACCACCTACTACAACCCCAGCCTGAAGAGTCGGATCTCCATCACTCGGGACACATCCAAGAACCAGTTCTACCTGCAGCTGAACTCTGTGACCACCGAGGACACCGCCACCTACTTCTGCGCCGGCGGCGCCAACAGCTTCGACTACTGGGGCCAGGGCACCACTCTCACTGTCTCCTCA
SEQ ID NO.10：
GACATCCAGATGACTCAGTCTCCAGCTTCACTGTCTGCATCTGTGGGCGAGACCGTGACCATCACCTGCGGCGCCGCCGAGAACATCTACGGCGCCCTGAACTGGTACCAGCGGAAACAGGAGAAGAGCCCTCAGCTCCTGATCTATGGTGCCACCAACCTGGCCGACGGCATGTCATCGAGGTTCAGTGGCAGTGGATCTGGTAGACAGTATAGCCTGAAGATCAGCAGCCTGCACCCCGACGACGTTGCAACGTATTACTGTCAAAATGTGCTGACCAACCCCTTCACCTTCGGCGGCGGCACCAAGCTGGAGATCAAA
EXAMPLE 5 construction of monoclonal antibody expression vectors and purification of expression
(1) Construction of expression vectors for monoclonal antibodies
According to the sequence determination result of the mouse monoclonal antibody, setting proper enzyme cutting sites, and respectively carrying out gene synthesis on heavy chain and light chain; the gene synthesis products were ligated into pcDNA3.4 expression vectors, respectively.
(2) Expression and purification of monoclonal antibodies
Expression of monoclonal antibodies: the procedure for cell transfection was the same as in (3) in example 1.
(3) Purification of monoclonal antibodies
(a) Harvesting cell culture solution: centrifuging to collect cell culture supernatant, and purifying antibody by using Protein A Beads;
(b) Antibody concentration determination was performed after purification was completed.
EXAMPLE 6 determination of monoclonal antibody molecular weight
Molecular weight of the monoclonal antibody was identified by SDS-PAGE electrophoresis, 5. Mu.g of each lane was loaded, and the molecular weight of the monoclonal antibody was analyzed by gel analysis after staining, using a standard series of known molecular weights as reference (marker), first electrophoresis at 90V for 20 min, then electrophoresis at 140V until the indicator was completely run out, removing the gel, staining with Coomassie blue. FIG. 1 is a SDS-PAGE result of a monoclonal antibody, wherein the heavy and light chains of the monoclonal antibody are mainly distributed at about 50 kDa and 25 kDa, as shown in FIG. 1, lane M in FIG. 1 1 Lane 1 is monoclonal antibody for marker.
EXAMPLE 7 detection of affinity Activity (potency) of monoclonal antibodies against helicobacter pylori by ELISA
The affinity activity is detected by the following steps:
helicobacter pylori antigen was diluted to 1 ng/. Mu.L with PBS and added to 96-well ELISA plates at 100. Mu.L per well, coated at 37℃for 2 h; the supernatant was discarded, the plate was washed 3 times with 0.01. 0.01M PBST to prepare a blocking solution containing 3% BSA, 100. Mu.L was added to each well, and blocking was performed at 37℃for 2 h; the supernatant was discarded, washed 5 times with PBST, the purified and concentrated antibody was subjected to gradient dilution at a concentration of 1:1000-fold to 1:2560000, wells were added with 100. Mu.L each, and incubated at 37℃for 1 h; discarding the antibody dilution, washing with PBST for 6 times, diluting HRP-labeled goat anti-mouse IgG (secondary antibody) with 1:5000 blocking solution, adding 100 μl of secondary antibody into each well, and incubating at 37deg.C for 1 h; discarding secondary anti-dilution, cleaning with PBST for 6 times, adding 100 μl of TMB into each well, and standing at 37deg.C for 15 min in dark; the reaction was stopped by adding 50. Mu.L of 1, M dilute sulfuric acid to each well, and the absorbance was measured at 450. 450 nm.
The detection result of the affinity activity of the monoclonal antibody is shown in figure 2, and under the condition that the OD value is kept unchanged, the larger the dilution multiple of the monoclonal antibody is, the stronger the binding capacity of the antigen-antibody is, the screened monoclonal antibody has stronger binding capacity to helicobacter pylori antigens, the potency to helicobacter pylori antigens is high, and the EC50 value reaches 0.003976 ng/mu L.
Example 8 detection of monoclonal antibody specificity by ELISA method
The specificity of the monoclonal antibodies was detected by cross-reaction, the steps of which are as follows:
coating the ELISA plate with Bacillus proteus, klebsiella pneumoniae, enteropathogenic Escherichia coli, salmonella enteritidis, salmonella typhimurium, staphylococcus aureus and Escherichia coli, wherein the coating amount of each hole is 50 ng; the monoclonal antibody was diluted to 10 ng/mL, added to each ELISA plate, 100. Mu.L was added to each well, and incubated at 37℃for 1 h; after washing, 100 μl of secondary antibody (HRP-labeled goat anti-mouse IgG) was added to each well and incubated at 37 ℃ for 0.5 h; after washing TMB was added and incubated at 37℃for 15 min, the reading was terminated. The cross reaction results are shown in figure 3, and the results show that the obtained monoclonal antibody does not cross react with other enteropathogenic bacteria, and has strong specificity.
In summary, the invention provides the helicobacter pylori resistant monoclonal antibody which is a murine monoclonal antibody, has good antigen specificity and high affinity, and has important application value in preparing helicobacter pylori detection products.
The applicant declares that the above is only a specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and it should be apparent to those skilled in the art that any changes or substitutions that are easily conceivable within the technical scope of the present invention disclosed by the present invention fall within the scope of the present invention and the disclosure.
Claims (9)
1. An anti-helicobacter pylori monoclonal antibody, characterized in that the heavy chain variable region of the antibody comprises a heavy chain CDR1 shown in SEQ ID NO.1, a heavy chain CDR2 shown in SEQ ID NO.2, and a heavy chain CDR3 shown in SEQ ID NO. 3; the light chain variable region of the antibody comprises a light chain CDR1 shown in SEQ ID NO.4, a light chain CDR2 shown in SEQ ID NO.5 and a light chain CDR3 shown in SEQ ID NO. 6.
2. The anti-helicobacter pylori monoclonal antibody according to claim 1, characterized in that the antibody comprises: a heavy chain variable region of an amino acid sequence as shown in SEQ ID NO. 7; and, a light chain variable region of an amino acid sequence shown as SEQ ID NO. 8.
3. The anti-helicobacter pylori monoclonal antibody according to any one of claims 1-2, characterized in that it further comprises any one or a combination of at least two of murine IgG1, igG2, igG3 or IgG4 constant regions, preferably murine IgG1 constant regions.
4. A nucleic acid molecule encoding the anti-helicobacter pylori monoclonal antibody according to any one of claims 1 to 3;
preferably, the nucleotide sequence encoding the heavy chain variable region of the anti-helicobacter pylori monoclonal antibody comprises the sequence shown in SEQ ID NO. 9; the nucleotide sequence of the light chain variable region of the anti-helicobacter pylori monoclonal antibody comprises a sequence shown in SEQ ID NO. 10.
5. An expression vector comprising the nucleic acid molecule of claim 4.
6. A host cell comprising at least one copy of the expression vector of claim 5 or having integrated into its genome the nucleic acid molecule of claim 4;
preferably, the host cell comprises a 293F cell or CHO cell.
7. A pharmaceutical composition comprising the anti-helicobacter pylori monoclonal antibody of any one of claims 1-3;
preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.
8. A kit for detecting helicobacter pylori, characterized in that the kit for detecting helicobacter pylori comprises the anti-helicobacter pylori monoclonal antibody according to any one of claims 1 to 3;
preferably, the kit for detecting helicobacter pylori further comprises any one or a combination of at least two of a positive control, a negative control, an antibody diluent, a chromogenic solution, a stop solution, a blocking solution and a washing solution.
9. Use of any one or a combination of at least two of the anti-helicobacter pylori monoclonal antibody of any one of claims 1-3, the nucleic acid molecule of claim 4, the expression vector of claim 5, the host cell of claim 6 or the pharmaceutical composition of claim 7 for the preparation of a medicament for the treatment of helicobacter pylori infection disease and/or a test product.
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