CN113075403B - Molecular marker and kit for diagnosing gastric cancer - Google Patents
Molecular marker and kit for diagnosing gastric cancer Download PDFInfo
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- CN113075403B CN113075403B CN202110305012.5A CN202110305012A CN113075403B CN 113075403 B CN113075403 B CN 113075403B CN 202110305012 A CN202110305012 A CN 202110305012A CN 113075403 B CN113075403 B CN 113075403B
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- 206010017758 gastric cancer Diseases 0.000 title claims abstract description 48
- 201000011549 stomach cancer Diseases 0.000 title claims abstract description 48
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- 238000001514 detection method Methods 0.000 claims abstract description 37
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- 238000005571 anion exchange chromatography Methods 0.000 claims description 10
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 9
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- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 claims description 3
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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
- 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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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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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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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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
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
- G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
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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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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention belongs to the technical field of kit preparation, and discloses a molecular marker for diagnosing gastric cancer and a kit, wherein the molecular marker is a segment of DNA sequence in MZF1 gene, and the DNA sequence is shown as SEQ ID No. 1; the molecular marker comprises a specific nucleotide sequence shown as SEQ ID No. 2; the kit for gastric cancer diagnosis comprises: ELISA plate, monoclonal antibody and immunomagnetic bead. The invention detects gastric cancer through the molecular markers for diagnosing and treating gastric cancer, has the characteristics of strong sensitivity and high specificity, can provide basis for monitoring gastric cancer patients' illness state and individual medication and early diagnosis of tumors, and can also provide an evaluation method for prognosis detection of drug treatment. The kit provided by the invention has the advantages of simple preparation method, high accuracy in gastric cancer detection by using the kit, short detection time and capability of providing assistance for diagnosis and prediction of gastric cancer.
Description
Technical Field
The invention belongs to the technical field of kit preparation, and particularly relates to a molecular marker for diagnosing gastric cancer and a kit.
Background
At present, gastric cancer (GASTRIC CANCER, GC) is one of the most common high-incidence tumors in China, and the mortality rate of the gastric cancer is in the front of various malignant tumors in the world and China. Due to the lack of effective detection means for early detection, most of patients at middle and late stages have lost their opportunity for surgery or are undergoing surgery after neoadjuvant chemotherapy. The clinical treatment of patients in middle and late stages is mainly chemotherapy, and the treatment mode can inhibit the tumor from deteriorating to a certain extent, but adverse reaction and pain caused by the toxic effect of the chemotherapy and the tolerance of patients in later stages to chemotherapy drugs bring about a series of biological and clinical problems. Therefore, the molecular pathway mechanism of tumor chemotherapy drug resistance is researched, so that molecular targets capable of inhibiting tumor cell proliferation and improving chemotherapy sensitivity are identified, a new idea is provided for accurate diagnosis and personalized treatment of tumors, and the method has important clinical significance and scientific value. However, the currently used gastric cancer diagnosis kit has the disadvantages of poor detection accuracy, long detection time, complex detection operation and difficulty in meeting the practical requirements.
Through the above analysis, the problems and defects existing in the prior art are as follows: the existing gastric cancer diagnosis kit has the defects of poor detection accuracy, long detection time, complex detection operation and difficulty in meeting the practical requirements.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a molecular marker and a kit for diagnosing gastric cancer.
The invention is realized in that a preparation method of a kit for diagnosing gastric cancer comprises the following steps:
Cloning a molecular marker into a eukaryotic expression vector, realizing the expression of proteins in mouse cells, purifying the proteins to obtain antigens containing the molecular marker, and immunizing the mice by using the antigens; cutting the spleen of the mouse, putting the mouse into a centrifuge tube for centrifugation, and removing supernatant; adding sterile normal saline into the precipitate for two times of cleaning to obtain treated B lymphocytes; taking myeloma cells, placing the myeloma cells into a centrifuge tube for centrifugation, and removing supernatant; adding sterile normal saline into the precipitate for two times of cleaning to obtain myeloma cells;
step two, mixing the treated B lymphocytes with myeloma cells, adding a fusion agent, placing the mixture in a centrifuge tube for centrifugation, and removing supernatant; adding PBS buffer solution into the precipitate, and carrying out water bath for 80-120 s in the water temperature of 37 ℃; then adding PBS buffer solution at 37 ℃ to shake lightly, and centrifuging for 3-5 min again to obtain fused hybridoma cells;
Step three, after the hybridoma cells appear, absorbing supernatant fluid, and detecting activity; selecting a proper hole according to the supernatant activity detection result, selecting a single fusion cell by adopting a flow cell sorting method, and performing first subcloning; sucking the supernatant after the first subcloning when the first subcloning cell mass grows to be obvious, and detecting the activity;
Step four, selecting a proper hole for secondary subcloning according to the supernatant activity detection result obtained in the step three, and expanding to a 24-pore plate for proliferation; sucking the supernatant after the second subcloning when the second subcloning cell mass grows to be obvious, and detecting the activity;
Step five, selecting proper holes to be expanded to 24 pore plates for expansion according to the supernatant activity detection result obtained in the step four; after the 24-pore plate cells grow to 80% of confluence rate, absorbing supernatant, and detecting activity; selecting proper holes to expand to a 25cm cell culture bottle according to the supernatant activity detection result for proliferation, freezing and preserving seeds to obtain a monoclonal antibody, and establishing a monoclonal antibody cell library;
Step six, monoclonal antibody is treated; the treated monoclonal antibody is subjected to first adjustment by using a pH adjusting solution, and the pH is adjusted to be 4.00-7.00; filtering, and carrying out secondary adjustment by using pH adjustment liquid, wherein the pH is adjusted to 8.00-9.00, so as to obtain the monoclonal antibody for adjusting the pH; carrying out anion exchange chromatography on the monoclonal antibody with the pH adjusted back to obtain a purified monoclonal antibody;
Step seven, using commercial carboxyl modified superparamagnetism microsphere as magnetic bead carrier, cleaning with 10-15 Mm MES buffer solution with pH of 5.00-6.00, and then mixing according to the formula of 1:1, adding 1.25M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution into the mixture in a volume ratio, and carrying out magnetic separation after light-shielding oscillation reaction for 25-35 min at room temperature to obtain activated magnetic beads; the purified monoclonal antibody was buffered with carbonate buffer according to 3:1, diluting the mixture in a volume ratio, and adding activated magnetic beads; adsorbing, washing, sealing and drying to obtain monoclonal antibody coated immunomagnetic beads;
diluting the immune magnetic beads coated by the monoclonal antibodies by using a sample diluent, adding 100 mu L/hole of the diluted immune magnetic beads to an ELISA plate, and magnetically separating and washing to obtain a detection reagent; and (3) dripping a sealing buffer solution into the hole of the enzyme-labeled plate where the detection reagent is positioned, wherein the dripping amount is 200 mu L/hole, and obtaining the kit for diagnosing gastric cancer.
In the first step, the molecular marker is a DNA sequence located in the MZF1 gene, and the DNA sequence is shown as SEQ ID No. 1.
Further, in the first step, the molecular marker comprises a specific nucleotide sequence shown as SEQ ID No. 2.
Further, in step one, the method for immunizing mice with the molecular marker comprises:
(1) Taking a plurality of Balb/c healthy mice of 6-8 weeks;
(2) Mixing an antigen containing a molecular marker with Freund's complete adjuvant in a volume ratio of 1:1, emulsifying to form a water-in-oil state, and performing subcutaneous multipoint injection on the mice to realize primary immunization;
(3) On day 14, mixing the antigen and Freund's incomplete adjuvant in a volume ratio of 1:1, emulsifying to form a water-in-oil state, and performing subcutaneous multipoint injection on the mice to realize secondary immunization;
(4) And (3) repeating the step (3) for three times with 14 days as a period to realize multiple immunization of the mice.
In the fifth step, the monoclonal antibody is IgA or IgG.
In the sixth step, the pH adjusting solution is any one of TrisHCl buffer, phosphate buffer, glycine-sodium hydroxide buffer, and sodium chloride-sodium hydroxide buffer.
Further, in the sixth step, the method for performing anion exchange chromatography on the monoclonal antibody with the pH adjusted back comprises the following steps:
(1) Pre-balancing the anion chromatographic column by using a pre-balancing buffer solution, and then balancing the anion chromatographic column by using a balancing buffer solution;
(2) Adding the monoclonal antibody with the pH adjusted back into an anion chromatographic column with good balance, and collecting an effluent A from 40 mAU; then washing the chromatographic column with the eluent, and collecting an effluent B before 40 mAU;
(3) Eluting the chromatographic medium with eluent, and washing the chromatographic column with distilled water and ethanol;
(4) Mixing the effluent A and the effluent B to obtain the monoclonal antibody after anion chromatography, and obtaining the purified monoclonal antibody.
Further, the pre-equilibrium buffer solution and the eluent are both high-salt Tris-HCl buffer solution, and the pH value of the high-salt Tris-HCl buffer solution is 8.00-10.00.
Another object of the present invention is to provide a kit for gastric cancer diagnosis prepared by using the method for preparing the kit for gastric cancer diagnosis, comprising: ELISA plate, monoclonal antibody and immunomagnetic bead.
Another object of the present invention is to provide a method for using a kit for gastric cancer diagnosis, comprising the steps of:
(1) Adding a diluted serum sample to be detected into an ELISA plate, and carrying out light-proof shaking incubation for 20-60 min at 37 ℃;
(2) Magnetic separation and washing, and then adding a monoclonal antibody mixed solution marked by horseradish peroxidase or alkaline phosphatase to obtain a compound;
(3) And (3) incubating for 20-60 min at 37 ℃ in a dark shaking way, transferring the complex taking the immunomagnetic beads as a liquid phase carrier to a new micro-pore plate, magnetically separating and washing the complex, and detecting and reading the detection result on an upper machine.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention detects gastric cancer through the molecular marker for diagnosing and treating gastric cancer and detecting gastric cancer, and the molecular marker has the characteristics of strong sensitivity and high specificity; the kit based on the molecular marker can carry out specific and quantitative detection on the DNA methylation level related to the MZF1 gene, provides a basis for monitoring the illness state and individual medication of gastric cancer patients and early diagnosis of tumors, and also provides an evaluation method for prognosis detection of the patients on drug treatment. The kit provided by the invention has the advantages of simple preparation method, high accuracy in gastric cancer detection by using the kit, simple detection method, short detection time and assistance in diagnosis and prediction of gastric cancer.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments of the present application will be briefly described below, and it is obvious that the drawings described below are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
Fig. 1 is a flowchart of a method for preparing a kit for gastric cancer diagnosis according to an embodiment of the present invention.
FIG. 2 shows B lymphocytes after treatment according to an embodiment of the present invention; and mixing the treated B lymphocytes with myeloma cells under the action of a fusion agent to obtain a flow chart of a method for fusing the hybridoma cells.
FIG. 3 is a flow chart of a method for screening, culturing and proliferating hybridoma cells to obtain monoclonal antibodies according to the embodiment of the invention.
FIG. 4 is a flow chart of a method for purifying a treated monoclonal antibody according to an embodiment of the present invention.
FIG. 5 is a flow chart of a method for anion exchange chromatography of a monoclonal antibody with a pH adjusted back according to an embodiment of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
Aiming at the problems existing in the prior art, the invention provides a molecular marker and a kit for diagnosing gastric cancer, and the invention is described in detail below with reference to the accompanying drawings.
The kit for diagnosing gastric cancer provided by the embodiment of the invention comprises the following components: ELISA plate, monoclonal antibody and immunomagnetic bead.
As shown in fig. 1, the preparation method of the kit for diagnosing gastric cancer provided by the embodiment of the invention comprises the following steps:
s101, cloning a molecular marker into a eukaryotic expression vector, realizing the expression of protein in a mouse cell, and purifying the protein to obtain an antigen containing the molecular marker;
S102, immunizing a mouse by using an antigen to obtain a treated B lymphocyte; mixing the treated B lymphocytes with myeloma cells under the action of a fusion agent to obtain fused hybridoma cells;
S103, screening, culturing and proliferating the hybridoma cells to obtain monoclonal antibodies, and performing humanization treatment on the monoclonal antibodies; purifying the treated monoclonal antibody, and coating the purified monoclonal antibody by using a magnetic bead carrier to obtain immune magnetic beads coated by the monoclonal antibody;
S104, diluting the immune magnetic beads coated by the monoclonal antibodies by using a sample diluent, adding 100 mu L/hole of the diluted immune magnetic beads to an ELISA plate, and magnetically separating and washing to obtain a detection reagent;
s105, dripping a sealing buffer solution into the hole of the enzyme-labeled plate where the detection reagent is positioned, wherein the dripping amount is 200 mu L/hole, and obtaining the kit for diagnosing gastric cancer.
In the step S101 provided by the embodiment of the invention, the molecular marker is a segment of DNA sequence in the MZF1 gene, and the DNA sequence is shown as SEQ ID No. 1.
In step S101 provided by the embodiment of the invention, the molecular marker comprises a specific nucleotide sequence shown as SEQ ID No. 2.
In step S102 provided in the embodiment of the present invention, a method for immunizing a mouse using an antigen containing a molecular marker includes:
(1) Taking a plurality of Balb/c healthy mice of 6-8 weeks;
(2) Mixing an antigen containing a molecular marker with Freund's complete adjuvant in a volume ratio of 1:1, emulsifying to form a water-in-oil state, and performing subcutaneous multipoint injection on the mice to realize primary immunization;
(3) On day 14, mixing the antigen and Freund's incomplete adjuvant in a volume ratio of 1:1, emulsifying to form a water-in-oil state, and performing subcutaneous multipoint injection on the mice to realize secondary immunization;
(4) And (3) repeating the step (3) for three times with 14 days as a period to realize multiple immunization of the mice.
As shown in fig. 2, in step S102 provided in the embodiment of the present invention, a method for obtaining a fused hybridoma cell by mixing a treated B lymphocyte and a myeloma cell under the action of a fusion agent, includes:
s201, cutting the spleen of the mouse, placing the mouse into a centrifuge tube for centrifugation, and removing supernatant; adding sterile normal saline into the precipitate for two times of cleaning to obtain treated B lymphocytes;
s202, taking myeloma cells, placing the myeloma cells into a centrifuge tube for centrifugation, and removing supernatant; adding sterile normal saline into the precipitate for two times of cleaning to obtain myeloma cells;
S203, mixing the treated B lymphocytes with myeloma cells, adding a fusion agent, placing the mixture in a centrifuge tube for centrifugation, and removing supernatant;
S204, adding PBS buffer solution into the precipitate, and carrying out water bath for 80-120S in the water temperature of 37 ℃; then adding PBS buffer solution at 37 ℃ to lightly shake, and centrifuging for 3-5 min again to obtain the fused hybridoma cells.
As shown in fig. 3, in step S103 provided in the embodiment of the present invention, a method for obtaining a monoclonal antibody by screening, culturing and proliferating hybridoma cells includes:
S301, after hybridoma cells appear, absorbing supernatant fluid, and detecting activity; selecting a proper hole according to the supernatant activity detection result, selecting a single fusion cell by adopting a flow cell sorting method, and performing first subcloning; sucking the supernatant after the first subcloning when the first subcloning cell mass grows to be obvious, and detecting the activity;
s302, selecting a proper hole for secondary subcloning according to the supernatant activity detection result obtained in the S301, and expanding to a 24-pore plate for proliferation; sucking the supernatant after the second subcloning when the second subcloning cell mass grows to be obvious, and detecting the activity;
S303, selecting proper holes to be expanded to 24 pore plates for expansion according to the supernatant activity detection result obtained in the S302; after the 24-pore plate cells grow to 80% of confluence rate, absorbing supernatant, and detecting activity; selecting proper holes to expand to a 25cm cell culture bottle according to the supernatant activity detection result for proliferation, freezing and preserving seeds to obtain monoclonal antibodies, and establishing a monoclonal antibody cell library.
In step S303 provided by the embodiment of the present invention, the monoclonal antibody is of IgA or IgG.
As shown in fig. 4, in step S103 provided in the embodiment of the present invention, a method for purifying a processed monoclonal antibody includes:
s401, performing first adjustment on the treated monoclonal antibody by using a pH adjustment liquid, and adjusting the pH to 4.00-7.00;
S402, filtering, and performing secondary adjustment by using pH adjustment liquid, wherein the pH is adjusted to 8.00-9.00, so as to obtain a monoclonal antibody for adjusting the pH;
S403, carrying out anion exchange chromatography on the monoclonal antibody with the pH adjusted back to obtain the purified monoclonal antibody.
In step S401 provided in the embodiment of the present invention, the pH adjusting solution is any one of TrisHCl buffer, phosphate buffer, glycine-sodium hydroxide buffer, and sodium chloride-sodium hydroxide buffer.
As shown in fig. 5, in step S403 provided in the embodiment of the present invention, a method for performing anion exchange chromatography on a monoclonal antibody with a pH adjusted back includes:
s501, pre-balancing the anion chromatographic column by using a pre-balancing buffer solution, and then balancing the anion chromatographic column by using a balancing buffer solution;
S502, adding the monoclonal antibody with the pH adjusted back into an anion chromatographic column with good balance, and collecting an effluent A from 40 mAU; then washing the chromatographic column with the eluent, and collecting an effluent B before 40 mAU;
S503, eluting the chromatographic medium by using eluent, and flushing the chromatographic column by using distilled water and ethanol;
S504, mixing the effluent A and the effluent B to obtain the monoclonal antibody after anion chromatography, and obtaining the purified monoclonal antibody.
In step S501 provided by the embodiment of the invention, the pre-equilibrium buffer solution and the eluent are both high-salt Tris-HCl buffer solution, and the pH value of the high-salt Tris-HCl buffer solution is 8.00-10.00.
In step S104 provided in the embodiment of the present invention, a method for coating a purified monoclonal antibody with a magnetic bead carrier to obtain a monoclonal antibody-coated immunomagnetic bead includes:
Commercial carboxyl modified superparamagnetism microsphere is used as a magnetic bead carrier, and is washed by 10-15Mm MES buffer solution with pH of 5.00-6.00 according to the following formula 1:1, adding 1.25M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution into the mixture in a volume ratio, and carrying out magnetic separation after light-shielding oscillation reaction for 25-35 min at room temperature to obtain activated magnetic beads;
Diluting monoclonal antibody with carbonate buffer solution, and adding activated magnetic beads; and (3) adsorbing, washing, sealing and drying to obtain the monoclonal antibody coated immunomagnetic beads.
The kit for diagnosing gastric cancer provided by the embodiment of the invention comprises the following components: ELISA plate, monoclonal antibody and immunomagnetic bead.
The application method of the kit for diagnosing gastric cancer provided by the embodiment of the invention comprises the following steps:
(1) Adding a diluted serum sample to be detected into an ELISA plate, and carrying out light-proof shaking incubation for 20-60 min at 37 ℃;
(2) Magnetic separation and washing, and then adding a monoclonal antibody mixed solution marked by horseradish peroxidase or alkaline phosphatase to obtain a compound;
(3) And (3) incubating for 20-60 min at 37 ℃ in a dark shaking way, transferring the complex taking the immunomagnetic beads as a liquid phase carrier to a new micro-pore plate, magnetically separating and washing the complex, and detecting and reading the detection result on an upper machine.
While the invention has been described with respect to what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but on the contrary, is intended to cover various modifications, equivalents, and alternatives falling within the spirit and scope of the invention.
Sequence listing
<110> College of long-term medical science
<120> Molecular marker and kit for gastric cancer diagnosis
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4000
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 1
ataaaagcac aaagttggat acaaaactgc ttagtgagct acaaagcgat acactggaca 60
gaaagcattt gcaacctatc aacctgtaca gggtagcata taactggaca gcatctggac 120
tcctaagttt tcctaacagt tttacctcgg attctccctg tctctcttcg tgctgtcatc 180
tttctctagg tcttcgaaaa ctgtcaatcc atcttactgt atctctaatt tctgtccctt 240
ttctcccttc ttgtttctgt ccctctccct tcttgtctac ctcttgagtc tggatatctg 300
catttcttgt ctctgtgaac ccattctgcg tttccttctt tgtgtctctt tcatctgcat 360
ccctttctga tctgcctctc tcttgctaac tgttctgaca cctgtttgcc cggccctgac 420
cctctgatcc actcagccct cttataactt ggacccgtgt ctctatgttt gttcacttat 480
tttcactgtc tctcctcgcg tcgagtctcc ggtcctccca gtctgtgagt ctgtctctct 540
gtctctctct ctcttcctcc cgccggtcct tcgtctctct ttgcccttga gtctctggtc 600
tctctccgtc tctcctcgct gtttctatcg atcggcatct ctctgcccca gtacccttcc 660
ccacctcttc atcccacatg ccccattgca ttttcaacgc ccgccgcgtc tccgggttac 720
ccaggcgatg tcgagtcgtg tccccaggtt accatggaga cgttcagggc ccgcccctcg 780
cccatgatca cagaggaacg tcatcgcgcc gcgcgcgcac gtacacacga agggcctgct 840
gactacatct gccggaaagc atcgggccaa gcctttctcc attttgcggt ctaggaagta 900
gcagaggccc cttcctgtag ggagttgcca tggagacgcg gtggggcacc gacggagttc 960
taatgacggc cgtgattggt gcaggatcct gctaatctca ggaaggcccg tagagaagtg 1020
aggaaaacgt ggtggggggc atgcgcgatc tggtaggcgg tgctgccgtc tgttgtacct 1080
gagaggcttg cgcatgccga cgcacggatt cgaggcgggg agcatgggaa gaagcggcca 1140
ggagtatgac ctgatcattg cgaccaccgc taggggaagg gaggagaggg tgtagaaacg 1200
gggacgaggg tgggggaagg gcaaggaggc gctcgagctg gtgcgcggag catcctggga 1260
gacgtagtcc agcgggaggg ggaagtcgaa gactgcgcgt gctcaggagc gcggagcggc 1320
ccgctgagcg cagaggtgag ggcggggtcc tcttaaccgg gagggctatc gtgtggacgg 1380
gggcggtggc tgcagactcg gggattccgg ctccccagtg agaccggaag cgcggggaac 1440
gaatccgggc agtccttgcg ggagcgccca gggctagagc gagaggcttg tcaatcacta 1500
ctcgttgcca cgacgacagg ctttgggagc cccgcccccg gttgcctagg cgtcgtctgg 1560
acctaccttg gcaccctgga gcggaagttg ccgcctggac ttctgggaag tgtatttcgg 1620
gaccctccgt aggtacctgc catcttggct atgacccaag tctctcccag ggtttcaggt 1680
tgacttggca ccttgaagag tggaggcttg gcccccagga tgaggcggcc ctcgaaatcg 1740
atgccaccca cgggggaatg attctgcggt gccatcccct tcataaacgc agattccttc 1800
ctaaagccgc agctgcagaa ggaacagcgc gaggagggag gtcagcccca cttgcaaatg 1860
gggaaacaag ttctcgaagg cacagtgatc tgaagatggg cggaatcaga gtctcgggac 1920
ttccttgcag gctcctgttc cggcagtctt ggagcctttg ccgccccccc acccattgcg 1980
aagctccccc atccccttcc aaccccagct aaatcaggaa gctgagatgg ggcattgcgt 2040
agtgtgatgt tgggcttaga agatggggct gagtagggag agagggtgct gcctgggagc 2100
tgagccatac aagtgactgc acaggttgac atggaggatt aggtggagtg aggcttccaa 2160
gcagggaggg gaatgatggt ggggcccaaa tgaggagcca catcgaagta gatgagagaa 2220
tagaaggtga agtaagggct ggcgttgggt agggggagac gccagcagtg atgctgatgc 2280
ccaggctgta ggtgtatagg tgccatccac ctggtaaaga gagagctgta gcgcaggaat 2340
gaggttgcac atgtagaaga agggaaggat acaggggaga gaagtgtctt ctagtcctaa 2400
aaaacagcct gtgggctggc atggtggaac aaacctgtaa gtcccagcac ttcgggaggt 2460
caaggtaaga ggatcatctg cttgagccca ggagttcaag aacagcctag gcaacatagt 2520
aagatcccat ccctacagaa aaattaagaa attagccgga tgtcgtggca cacacctgtt 2580
gtctcagcta cttgggaggc cgatctctgg agcccgggag gtcaagtctg cagtgacgca 2640
tgatctcgcc actgcacttc agcctgggca actgactgac agcatgtctc aaagaaaaga 2700
aaaaaaagcc tgtgtttgga gcagggagac tgtcagagac tttgcacagg cccaagcgga 2760
gagaagacca gggatacagg tggctaaggg agggcttcca gctgcataag tctgtgggtt 2820
gtagatggcc tttctttgcc tgtttcctca tctgccttcc acatggggta ggtctgagga 2880
tcaaacggta cagcatagac taggcagtga agcattcact gtactccttt tgcaacagcc 2940
tcatcctatt tcttcaccag gggcagacac tggcctcaga tacctgacct ggtaccctct 3000
atgaggcctg cggtgctggg ctccccagac cgagcacccc cagaagatga ggggcctgtc 3060
atggtgaagc tagaggactc tgaggaggag ggtgaggctg ccttatggga cccaggccct 3120
gaagctgcac gcctgcgttt ccggtgcttc cgctatgagg aggccacagg gccccaagag 3180
gccctggccc agctccgaga gctgtgtcgc cagtggctgc gtccagaggt acgctccaag 3240
gagcagatgc tggagctgtt ggtgctggag cagttcctgg gcgcactgcc ccctgagatc 3300
caggcccgtg tgcaggggca gcggccaggc agccccgagg aggctgctgc cctagtagat 3360
gggctgcgcc gggagccggg cggaccccgg agatgggtga gtgagtgtcc acgagtgggg 3420
ttcaggactg gagcatcatc cagctcggcc tcaccctggg cagcaccacg ctttgccaga 3480
cacacgttct cccttgtggt acagaagtgg ccacctgcaa cagagaccct gtgcaaggct 3540
gcccagggca gagggattcc agggccagac tccccagccc attcctgcat cacctgcagt 3600
cacgtggaca taggagctgc aggctagggg aatatgggga aggtactgga aggccacgat 3660
gtcagagcag gggagggact gcaggtggtc ccttcattct ttgctcaggg cctaggggag 3720
gtaggtcctt ggaggaccta gtcagtaggt atttacaagg caggcccctg ggatcctaca 3780
gtgggatggg cacccacaaa ccccaggttg cggccagcct tactctctgg taggacttct 3840
gatggtgggg gcacctcccc aggtcacagt ccaggtgcag ggccaggagg tcctatcaga 3900
gaagatggag ccctccagtt tccagcccct acctgaaact gagcctccaa ctccagagcc 3960
tgggcccaag acacctccta ggactatgca ggaatcacca 4000
<210> 2
<211> 2000
<212> DNA
<213> Artificial sequence (ARTIFICIAL SEQUENCE)
<400> 2
cactggacag aaagcatttg caacctatca acctgtacag ggtagcatat aactggacag 60
catctggact cctaagtttt cctaacagtt ttacctcgga ttctccctgt ctctcttcgt 120
gctgtcatct ttctctaggt cttcgaaaac tgtcaatcca tcttactgta tctctaattt 180
ctgtcccttt tctcccttct tgtttctgtc cctctccctt cttgtctacc tcttgagtct 240
ggatatctgc atttcttgtc tctgtgaacc cattctgcgt ttccttcttt gtgtctcttt 300
catctgcatc cctttctgat ctgcctctct cttgctaact gttctgacac ctgtttgccc 360
ggccctgacc ctctgatcca ctcagccctc ttataacttg gacccgtgtc tctatgtttg 420
ttcacttatt ttcactgtct ctcctcgcgt cgagtctccg gtcctcccag tctgtgagtc 480
tgtctctctg tctctctctc tcttcctccc gccggtcctt cgtctctctt tgcccttgag 540
tctctggtct ctctccgtct ctcctcgctg tttctatcga tcggcatctc tctgccccag 600
tacccttccc cacctcttca tcccacatgc cccattgcat tttcaacgcc cgccgcgtct 660
ccgggttacc caggcgatgt cgagtcgtgt ccccaggtta ccatggagac gttcagggcc 720
cgcccctcgc ccatgatcac agaggaacgt catcgcgccg cgcgcgcacg tacacacgaa 780
gggcctgctg actacatctg ccggaaagca tcgggccaag cctttctcca ttttgcggtc 840
taggaagtag cagaggcccc ttcctgtagg gagttgccat ggagacgcgg tggggcaccg 900
acggagttct aatgacggcc gtgattggtg caggatcctg ctaatctcag gaaggcccgt 960
agagaagtga ggaaaacgtg gtggggggca tgcgcgatct ggtaggcggt gctgccgtct 1020
gttgtacctg agaggcttgc gcatgccgac gcacggattc gaggcgggga gcatgggaag 1080
aagcggccag gagtatgacc tgatcattgc gaccaccgct aggggaaggg aggagagggt 1140
gtagaaacgg ggacgagggt gggggaaggg caaggaggcg ctcgagctgg tgcgcggagc 1200
atcctgggag acgtagtcca gcgggagggg gaagtcgaag actgcgcgtg ctcaggagcg 1260
cggagcggcc cgctgagcgc agaggtgagg gcggggtcct cttaaccggg agggctatcg 1320
tgtggacggg ggcggtggct gcagactcgg ggattccggc tccccagtga gaccggaagc 1380
gcggggaacg aatccgggca gtccttgcgg gagcgcccag ggctagagcg agaggcttgt 1440
caatcactac tcgttgccac gacgacaggc tttgggagcc ccgcccccgg ttgcctaggc 1500
gtcgtctgga cctaccttgg caccctggag cggaagttgc cgcctggact tctgggaagt 1560
gtatttcggg accctccgta ggtacctgcc atcttggcta tgacccaagt ctctcccagg 1620
gtttcaggtt gacttggcac cttgaagagt ggaggcttgg cccccaggat gaggcggccc 1680
tcgaaatcga tgccacccac gggggaatga ttctgcggtg ccatcccctt cataaacgca 1740
gattccttcc taaagccgca gctgcagaag gaacagcgcg aggagggagg tcagccccac 1800
ttgcaaatgg ggaaacaagt tctcgaaggc acagtgatct gaagatgggc ggaatcagag 1860
tctcgggact tccttgcagg ctcctgttcc ggcagtcttg gagcctttgc cgccccccca 1920
cccattgcga agctccccca tccccttcca accccagcta aatcaggaag ctgagatggg 1980
gcattgcgta gtgtgatgtt 2000
Claims (9)
1. A method for preparing a kit for diagnosing gastric cancer, which is characterized by comprising the following steps:
Cloning a molecular marker into a eukaryotic expression vector, realizing the expression of proteins in mouse cells, purifying the proteins to obtain antigens containing the molecular marker, and immunizing the mice by using the antigens; cutting the spleen of the mouse, putting the mouse into a centrifuge tube for centrifugation, and removing supernatant; adding sterile normal saline into the precipitate for two times of cleaning to obtain treated B lymphocytes; taking myeloma cells, placing the myeloma cells into a centrifuge tube for centrifugation, and removing supernatant; adding sterile normal saline into the precipitate for two times of cleaning to obtain myeloma cells;
step two, mixing the treated B lymphocytes with myeloma cells, adding a fusion agent, placing the mixture in a centrifuge tube for centrifugation, and removing supernatant; adding PBS buffer solution into the precipitate, and carrying out water bath for 80-120 s in the water temperature of 37 ℃; then adding PBS buffer solution at 37 ℃ to shake lightly, and centrifuging for 3-5 min again to obtain fused hybridoma cells;
Step three, after the hybridoma cells appear, absorbing supernatant fluid, and detecting activity; selecting a proper hole according to the supernatant activity detection result, selecting a single fusion cell by adopting a flow cell sorting method, and performing first subcloning; sucking the supernatant after the first subcloning when the first subcloning cell mass grows to be obvious, and detecting the activity;
Step four, selecting a proper hole for secondary subcloning according to the supernatant activity detection result obtained in the step three, and expanding to a 24-pore plate for proliferation; sucking the supernatant after the second subcloning when the second subcloning cell mass grows to be obvious, and detecting the activity;
Step five, selecting proper holes to be expanded to 24 pore plates for expansion according to the supernatant activity detection result obtained in the step four; after the 24-pore plate cells grow to 80% of confluence rate, absorbing supernatant, and detecting activity; selecting proper holes to expand to a 25cm cell culture bottle according to the supernatant activity detection result for proliferation, freezing and preserving seeds to obtain a monoclonal antibody, and establishing a monoclonal antibody cell library;
Step six, monoclonal antibody is treated; the treated monoclonal antibody is subjected to first adjustment by using a pH adjusting solution, and the pH is adjusted to be 4.00-7.00; filtering, and carrying out secondary adjustment by using pH adjustment liquid, wherein the pH is adjusted to 8.00-9.00, so as to obtain the monoclonal antibody for adjusting the pH; carrying out anion exchange chromatography on the monoclonal antibody with the pH adjusted back to obtain a purified monoclonal antibody;
Step seven, using commercial carboxyl modified superparamagnetism microsphere as magnetic bead carrier, cleaning with 10-15 Mm MES buffer solution with pH of 5.00-6.00, and then mixing according to the formula of 1:1, adding 1.25M 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution into the mixture in a volume ratio, and carrying out magnetic separation after light-shielding oscillation reaction for 25-35 min at room temperature to obtain activated magnetic beads; the purified monoclonal antibody was buffered with carbonate buffer according to 3:1, diluting the mixture in a volume ratio, and adding activated magnetic beads; adsorbing, washing, sealing and drying to obtain monoclonal antibody coated immunomagnetic beads;
Diluting the immune magnetic beads coated by the monoclonal antibodies by using a sample diluent, adding 100 mu L/hole of the diluted immune magnetic beads to an ELISA plate, and magnetically separating and washing to obtain a detection reagent; dripping a sealing buffer solution into the hole of the enzyme-labeled plate where the detection reagent is positioned, wherein the dripping amount is 200 mu L/hole, so as to obtain a kit for diagnosing gastric cancer;
In the first step, the molecular marker is a DNA sequence located in the MZF1 gene, and the DNA sequence is shown as SEQ ID No. 1.
2. The method for preparing a kit for diagnosing gastric cancer according to claim 1, wherein in the first step, the molecular marker comprises a specific nucleotide sequence as shown in SEQ ID No. 2.
3. The method for preparing a kit for diagnosing gastric cancer according to claim 1, wherein in the first step, the method for immunizing a mouse with the molecular marker comprises the following steps:
(1) Taking a plurality of Balb/c healthy mice of 6-8 weeks;
(2) Mixing an antigen containing a molecular marker with Freund's complete adjuvant in a volume ratio of 1:1, emulsifying to form a water-in-oil state, and performing subcutaneous multipoint injection on the mice to realize primary immunization;
(3) On day 14, mixing the antigen and Freund's incomplete adjuvant in a volume ratio of 1:1, emulsifying to form a water-in-oil state, and performing subcutaneous multipoint injection on the mice to realize secondary immunization;
(4) And (3) repeating the step (3) for three times with 14 days as a period to realize multiple immunization of the mice.
4. The method for preparing a kit for diagnosing gastric cancer according to claim 1, wherein in the fifth step, the monoclonal antibody is IgA or IgG.
5. The method for preparing a kit for diagnosing gastric cancer according to claim 1, wherein in the sixth step, the pH adjusting solution is any one of TrisHCl buffer, phosphate buffer, glycine-sodium hydroxide buffer, and sodium chloride-sodium hydroxide buffer.
6. The method for preparing a kit for diagnosing gastric cancer according to claim 1, wherein in the sixth step, the method for subjecting the monoclonal antibody having the pH adjusted back to anion exchange chromatography comprises the steps of:
(1) Pre-balancing the anion chromatographic column by using a pre-balancing buffer solution, and then balancing the anion chromatographic column by using a balancing buffer solution;
(2) Adding the monoclonal antibody with the pH adjusted back into an anion chromatographic column with good balance, and collecting an effluent A from 40 mAU; then washing the chromatographic column with the eluent, and collecting an effluent B before 40 mAU;
(3) Eluting the chromatographic medium with eluent, and washing the chromatographic column with distilled water and ethanol;
(4) Mixing the effluent A and the effluent B to obtain the monoclonal antibody after anion chromatography, and obtaining the purified monoclonal antibody.
7. The method for preparing a kit for diagnosing gastric cancer according to claim 6, wherein the pre-equilibration buffer and the eluent are both high-salt Tris-HCl buffer, and the pH value of the high-salt Tris-HCl buffer is 8.00-10.00.
8. A kit for gastric cancer diagnosis prepared by using the method for preparing the kit for gastric cancer diagnosis according to any one of claims 1 to 7, characterized in that the kit for gastric cancer diagnosis comprises: ELISA plate, monoclonal antibody and immunomagnetic bead.
9. A method of using the kit for gastric cancer diagnosis according to claim 8, characterized in that the method of using the kit for gastric cancer diagnosis comprises the steps of:
(1) Adding a diluted serum sample to be detected into an ELISA plate, and carrying out light-proof shaking incubation for 20-60 min at 37 ℃;
(2) Magnetic separation and washing, and then adding a monoclonal antibody mixed solution marked by horseradish peroxidase or alkaline phosphatase to obtain a compound;
(3) And (3) incubating for 20-60 min at 37 ℃ in a dark shaking way, transferring the complex taking the immunomagnetic beads as a liquid phase carrier to a new micro-pore plate, magnetically separating and washing the complex, and detecting and reading the detection result on an upper machine.
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| WO2010063138A1 (en) * | 2008-12-01 | 2010-06-10 | 苏州思坦维生物技术有限公司 | A monoclonal antibody specifically binding to vegf and the hybridoma secreting same and uses thereof |
| CN102645537A (en) * | 2012-04-26 | 2012-08-22 | 北京美康生物技术研究中心 | Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application |
| CN106405116A (en) * | 2016-12-06 | 2017-02-15 | 江西惠肽生物科技有限公司 | Kit for gastric cancer diagnosis and inspection method thereof |
| CN107085110A (en) * | 2017-05-31 | 2017-08-22 | 江西乐成生物医疗有限公司 | One kind is used for thyroid cancer external diagnosis reagent case and its detection method |
| CN109576364A (en) * | 2018-10-22 | 2019-04-05 | 北京交通大学 | Molecular marked compound and kit for diagnosing gastric cancer, chemotherapy and prognosis detection |
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| WO2010063138A1 (en) * | 2008-12-01 | 2010-06-10 | 苏州思坦维生物技术有限公司 | A monoclonal antibody specifically binding to vegf and the hybridoma secreting same and uses thereof |
| CN102645537A (en) * | 2012-04-26 | 2012-08-22 | 北京美康生物技术研究中心 | Latex enhanced turbidimetric immunoassay kit for diagnosing gastric diseases or gastric cancer, preparation method thereof and application |
| CN106405116A (en) * | 2016-12-06 | 2017-02-15 | 江西惠肽生物科技有限公司 | Kit for gastric cancer diagnosis and inspection method thereof |
| CN107085110A (en) * | 2017-05-31 | 2017-08-22 | 江西乐成生物医疗有限公司 | One kind is used for thyroid cancer external diagnosis reagent case and its detection method |
| CN109576364A (en) * | 2018-10-22 | 2019-04-05 | 北京交通大学 | Molecular marked compound and kit for diagnosing gastric cancer, chemotherapy and prognosis detection |
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