CN110218251A - Anti- MSH2 protein monoclonal antibody, cell line and its preparation method and application - Google Patents
Anti- MSH2 protein monoclonal antibody, cell line and its preparation method and application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/577—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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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- C07—ORGANIC CHEMISTRY
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- C07K2317/00—Immunoglobulins specific features
- 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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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
- C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
- G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
Abstract
The present invention relates to field of biological detection, a kind of anti-MSH2 protein monoclonal antibody is provided, the amino acid sequence of heavy chain and light chain variable region is amino acid sequence shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.Inventor additionally provides the preparation method of anti-MSH2 protein monoclonal antibody, chooses 411-424 albumen of people MSH2 amino acid and is coupled with KLH, as immunogene.Inventor additionally provides the hybridoma cell line of one plant of anti-MSH2 albumen of secretion, and the cell line is mouse hybridoma cell system 1B11-14-16, deposit number are as follows: CGMCC NO.17405.Anti- MSH2 protein monoclonal antibody has high specific, sensibility, and the cell of MSH2 albumen can be expressed with specific recognition, is suitable for immunology detection, especially immunohistochemistry and detects.
Description
Technical field
The present invention relates to field of biological detection, in particular to anti-MSH2 protein monoclonal antibody, cell line and its preparation side
Method and application.
Background technique
DNA mismatch repair system (MMR) is made of the enzyme molecule of special DNA plerosis base mispairing.Studies have shown that MMR
The mutation of gene and (or) afunction are related to the generation of kinds of tumors, such as colorectal cancer, cutaneum carcinoma, oophoroma, intrauterine
Film cancer etc..MSH2 (MutS homolog 2) gene be earliest discovery, be also scholars study the focus that is focused when MMR gene it
One, it is " house-keeping gene " in MMR system, has the function of identifying mispairing, the cDNA of long 3111bp contains opening for 2802bp
Reading frame is put, contains 934 amino acid in the albumen of coding.MSH6 is one of the important member that family is repaired in mispairing, DNA overall length
23806bp, cDNA long 4200bp, main function are to correct base mispairing and small fragment insertion and missing.When mispairing occurs for DNA
When, MSH6 albumen and MSH2 protein binding generate can to the MutS- α heterodimer that mismatch site is identified, the dimer with
Mismatch site starts mispairing repair process after combining, to guarantee microsatellite stability, by the spontaneous mutation rate of genes within cells
Control makes the stability of genome heredity and authenticity be protected in reduced levels.
MSH2 albumen almost comes across all colon normal mucosas, and positive rate is up to 94%.Positive products are positioned in core, portion
Cell is divided to occur endochylema dyeing simultaneously.Positive cell is distributed in mucous membrane holostrome, more intensive to arrange in 1/3-2/3 under gland nest.
The expression of accidental MSH2 albumen in cup cell.MSH2 albumen also express Activity of Intratumoral Fibroblasts inthe, vascular endothelial cell and
It is in caryogram or caryoplasm type, but expression is less in thick liquid cell on lymphocyte.
The mutation of MSH2 gene is related with microsatellite instability and certain cancers, and research finds that MSH2 gene protein is positive
Expression rate is gradually decreased from normal tissue, adenoma atypical hyperplasia, adenoma canceration to gland cancer, prompts to have had in adenoma stage
The mutation or dysfunction of the gene of MMR, and with the increase of its grade malignancy, the frequency of mutation of MMR gene also gradually rises.Table
The mutation of bright MMR gene, the mutation of especially MSH2 may be one of the earliest events that colorectal cancer occurs.Wherein heredity is non-
A kind of colorectal cancer of specific type of polyp characteristic of disease colorectal cancer (HNPCC).Hereditary nonpolyposis colorectal cancer
(HNPCC), sometimes referred to as Lynch syndrome, it is hereditary in a manner of autosomal dominant inheritance, wherein being only mutated mismatch repair gene
One copy heredity be just enough to cause disease phenotype, account for about colorectal cancer sum 15%~18%, it is straight to account for familial knot
The 50% of intestinal cancer.The mutation of MSH2 gene accounts for the 40% of disease related gene change, and mutation relevant to HNPCC is widely distributed
It in all structural domains of MSH2, and is the main reason for MLH1 is mutated.In addition MSH2 and other mismatch repair genes is prominent
Change also results in DNA damage and does not repair, and the frequency of mutation is caused to increase.
Summary of the invention
Inventor provide a kind of anti-MSH2 protein monoclonal antibody, the monoclonal antibody heavy and light chain variable region
Amino acid sequence is amino acid sequence shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.
Further, the monoclonal antibody heavy and chain variable region amino acid sequence be respectively SEQ ID NO.4 and
Coded by nucleotide sequence shown in SEQ ID NO.5.
Further, the monoclonal antibody specificity identifies MSH2 albumen.
Further, amino acid sequence shown in SEQ ID NO.1 in the monoclonal antibody specificity identification.
Further, the monoclonal antibody is generated by the hybridoma cell line that deposit number is CGMCC NO.17405.Institute
State cell strain be mouse hybridoma cell system 1B11-14-16, classification naming are as follows: mouse hybridoma cell system, the cell line in
In China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, address is court, Beijing on 03 21st, 2019
No. 3 Institute of Microorganism, Academia Sinica, institute of positive area's North Star West Road 1.
Further, the anti-MSH2 albumen is mouse IgG2bHypotype monoclonal antibody.
Inventor additionally provides a kind of preparation method of anti-MSH2 protein monoclonal antibody, chooses MSH2 Argine Monohydrochloride sequence
The 919th is arranged to add sulfydryl modification in its N-terminal to the 934th amino acids and carry out coupling as immunogene with carrier protein KLH.
Inventor additionally provides the hybridoma cell line of one plant of anti-MSH2 albumen of secretion, and the cell strain is Mouse Hybridoma Cells
Cell line 1B11-14-16, the cell line are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center,
Deposit number are as follows: CGMCC NO.17405.
Inventor also provides any of the above-described anti-MSH2 protein monoclonal antibody, in the detection of MSH2 protein immunization
Purposes.
Further, the immune detection includes immunohistochemical method, Western blot and enzyme-linked immunization.
It is different from the prior art, structure, antigenicity, group ammonification of the above-mentioned technical proposal according to MSH2 albumen in conjunction with DNA
The hydrophilic and hydrophobic and secondary structure of base acid, selection, which has, is different from other similar albumen, suitable length and with special antigen
919-934 albumen of amino acid sequence (SEQ ID No.1) of property, region is as Antigenic Peptide.Sulfydryl modification is added simultaneously in N-terminal
It is coupled KLH, as immunogen C (KLH-NSFVNEIISRIKVTT-Cys).Mouse is immunized, through cell fusion, screening and
Subclone obtains the monoclonal cell system 1B11-14-16 of the anti-MSH2 protein monoclonal antibody of efficient secretion, and by the cell
The secreted anti-MSH2 protein monoclonal antibody of system.The antibody that this programme obtains has high specific, sensibility, can specificity
The cell of recognition expression MSH2 albumen is suitable for immunology detection, especially immunohistochemistry and detects.
Detailed description of the invention
The polyacrylamide gel electrophoresis figure of Fig. 1: MSH2 monoclonal antibody after purification.
The western blot figure of Fig. 2: MSH2 monoclonal antibody detection people PD-1 albumen.
Fig. 3: for Colorectal Carcinoma immunohistochemical staining result figure, (MSH2 that a left side is 1B11-14-16, the right side are commercially available
MSH2)。
Fig. 4: for Bladder Cancer immunohistochemical staining result figure, (MSH2 that a left side is 1B11-14-16, the right side are commercially available
MSH2)。
Fig. 5: for tonsil immunohistochemical staining result figure, (MSH2 that a left side is 1B11-14-16, the right side are commercially available
MSH2)。
Fig. 6: for appendix tissue immunohistochemical staining result figure (MSH2 that a left side is 1B11-14-16, the right side are commercially available MSH2).
Specific embodiment
The preparation of the recombination MSH2 protein fragments of embodiment 1
One, gene cloning
From the MSH2 protein sequence of selection number P43246 in Uniprot database (http://www.uniprot.org)
As standard sequence.According to its structure in conjunction with DNA, antigenicity, the hydrophilic and hydrophobic and secondary structure for forming amino acid, choosing
Select be different from other similar albumen, suitable length and have special antigenic region as Antigenic Peptide.Selected MSH2 ammonia
Base acid sequence the 919th to the 934th bit sequence, corresponding nucleotides sequence is classified as SEQ ID No.1.Synthesize the above MSH2 egg
It is white, its N-terminal addition sulfydryl modification and and carrier protein KLH, improve its immunogenicity, it is spare as immunogene.
The foundation of 2 1B11-14-16 hybridoma cell line of embodiment
One, it is immunized
MSH2 albumen-KLH the conjugate obtained in embodiment 1 is diluted to 1mg/mL, then with it is isometric completely not
Family name's adjuvant (CFA, Sigma company) mixing and emulsifying carries out the Balb/c mouse (being purchased from Foochow Wu Shi experimental animal) of 18-20g
Abdomen injection is immune, and injection dosage is 50 μ g/.Hereafter every 14 days booster immunizations are primary, and antigen uses Freund non-fully adjuvant
(IFA, Sigma company) emulsification, dosage are 25 μ g/.2nd booster immunization
14 days how anti-potency with anti-immunity original in indirect ELISA (wavelength 450nm) detection mice serum afterwards, potency highest
Mouse it is immune with tail vein injection impact, antigen is mixed with PBS solution, dosage be 50 μ g/ only.
Two, cell fusion
Mouse boosting cell suspension up to standard is immunized in sterile preparation, with murine myeloma cell sp2/0 (ATCC) with 5:1 ratio
Mixing, 1000rpm are centrifuged 10min, and after abandoning supernatant, the PEG (Sigma that 1mL is preheated to 37 DEG C is added from slow to fast in 1 minute
Company) solution, it needs gently to rotate centrifuge tube in adition process, comes into full contact with cell with PEG.After being stored at room temperature 90s, in 2min
Serum-free DMEM (Hyclone company) culture medium that 4mL is preheated to 37 DEG C is added from slow to fast, is added in subsequent 2min
10mL preheats plasma-free DMEM medium, adds remaining preheating plasma-free DMEM medium in last 2min, is settled to 50mL,
Entire adition process needs to slowly shake centrifuge tube, it is ensured that is uniformly mixed, mitigates the injury to cell.After being stored at room temperature 10min
It is centrifuged (1000rpm, 5min), abandons supernatant, cell is resuspended with 10-20mLHAT (Sigma company) culture medium, and with HAT culture medium
It is diluted to final concentration of 0.5 × 106Cells/mL, all solution are transferred in 96 orifice plates, 200 holes μ L/, and are marked.It will
96 well culture plates are carefully transferred to 37 DEG C, cultivate in 5%CO2 incubator.The growth conditions for inspecting periodically cell and potentially dirt
Dye, pays attention to opening and closing incubator less as far as possible, it is ensured that culture environment is stablized.The 5th day after fusion, HAT culture medium is added into culture plate,
50 holes μ L/.
Three, ELISA screens positive hybridoma cell
When fused cell diameter it is long to about 1-2mm when, draw 50-200 μ L culture solution supernatant and carry out cell sieve for the first time
Choosing (ELISA, IHC-P and other methods detection), while HAT culture medium is added to 200 μ L into culture hole.ELISA detection should
Culture solution supernatant, the culture hole inner cell culture solution that will test to obtain positive findings are fully transferred to 24 well culture plates, add HT
Culture medium, the hole 2mL/ are cultivated 3 days.
Repeat screening 24 orifice plates in each cell strain, reject be not positive findings culture hole cell, obtain positive findings compared with
Good culture hole cell.The positive hole cell that these 24 well culture plates obtain is subjected to subclone screening using limiting dilution assay,
Limiting dilution assay is obtained to be transferred to CO in cell liquid 96 well culture plates of addition2It is incubator culture 11 days, straight to clone cell
When diameter is 1-2mm, repeat cell screening.According to testing result, each subcloned cells strain, selection 4 are well-grown
Monoclonal positive culture hole, and be transferred in 24 orifice plates and continue to cultivate.Clone in 24 orifice plates is screened after a period of time again to obtain
Positive colony cell strain, as secrete the hybridoma cell strain 1B11-14-16 of specific monoclonal antibody.This cell strain is turned
Enter to be expanded to logarithmic growth phase conservation in T-75 culture bottle or carries out subsequent experimental.
3 ascites of embodiment induces method preparation monoclonal antibody
One, prepared by ascites
It will be washed and be resuspended with serum free medium after cell strain culture to logarithmic growth phase, count 2 × 106A/mL.It is outstanding
10 days BALB/C mices of paraffin oil sensitization are used in floating cell solution intraperitoneal injection in advance, and every mouse injects 0.5mL.It raises small
Mouse started to acquire ascites after 7 days.The ascites of collection is centrifuged 10min in 4 DEG C, 8000rpm, draws supernatant solution and is collected in centrifuge tube
In, as ascites, 4 DEG C or -20 DEG C preservations.
Two, the purifying of monoclonal antibody
With rProtein A sepharose Fast Flow (GE company) affinity column antibody purification master from ascites
It is divided into: 1. fills column, the ProteinA filler of purchase is loaded in gravitational stratification column in right amount with equilibration buffer (0.1M Tris
Solution, pH7.0) it rinses to balance；2. loading will be added in the chromatographic column installed, control by the ascites of 0.22 μm of membrane filtration
1 drop/sec of flow velocity processed；3. balancing, Equilibration buffer wash to balance is used after upper complete sample liquid；4. eluting, elution buffer is added
(0.1M citric acid solution, pH4.5) rinses pillar and collects eluent；5. regenerating, equilibration buffer punching is added after the completion of elution
Pillar is washed to balancing, the 20% ethyl alcohol flushing of 2 times of column volumes is placed on 4 DEG C of preservations.
Antibody purity is finally identified using SDS-PAGE method, such as the polyacrylamide gel electricity of Fig. 1 MSH2 monoclonal antibody
It swims shown in figure, antibody purity reaches 95% or more.Ultraviolet micro-spectrophotometer method measures antibody concentration, and concentration reaches 2.7mg/
ML or more.
4 monoclonal antibody CHARACTERISTICS IDENTIFICATION of embodiment
One, subtype identification
Cell conditioned medium is diluted to 1 μ g/mL coated elisa plate, every hole adds 100 μ L, and 4 DEG C of coatings overnight, are emptied liquid, use
200 μ L confining liquids (the PBS-T solution containing 2%BSA) is added in PBS (PBS-T) containing 0.05%Tween board-washing 3 times, every hole, and 37
DEG C be incubated for 1h.It is emptied liquid, is cleaned 3 times with PBS-T.Every hole is added on the 0.1mL hybridoma cell strain culture solution for diluting 5 times
Clearly, 37 DEG C of incubation 1h.It is emptied liquid, is cleaned 3 times with PBS-T.With confining liquid 1:400 dilution HRP label sheep anti mouse (κ, λ,
IgM, IgG1, IgG2b, IgG2b, IgG3, IgA) and antibody (Southern Biotech company), every hole is separately added into 0.1mL, and 37
DEG C be incubated for 1h.It is emptied liquid, is cleaned 3 times with PBS-T.Every hole adds 100 μ LTMB (Huzhou Yingcheng Biological Technology Co., Ltd.) substrates
(the isometric mixed solution of A, B) develops the color, and reacts at room temperature 15min, it is anti-that 50 μ L 1N HCl solution color development stoppings are added in every hole
It answers, then the OD value under microplate reader measurement 450nm wavelength.The results show that monoclonal antibody of the present invention is IgG2bType source of mouse Dan Ke
Grand antibody.
Two, affinity costant measures
It is coated with MSH2 albumen, peridium concentration is 100 μ g/ml, and 100 holes μ l/, overnight, PBS-T is washed 3 times 4 DEG C of coatings.Every hole
37 DEG C of 200 μ l confining liquid closing 1h, PBS-T is added to wash 3 times.The monoclonal antibody purified in embodiment 4 is diluted to following concentration
(unit: ng/mL): 2000,500,125,62.5,31.25,15.625,3.125,0.625,37 DEG C of incubations 1h, PBS-T wash 3
It is secondary.The sheep anti mouse secondary antibody 1:5000 dilution of HRP label, 100 μ l of every hole, 37 DEG C of incubation 1h, PBS-T are washed 3 times.100 μ are added in every hole
L TMB (Huzhou Yingcheng Biological Technology Co., Ltd.) developing solution, develop the color 13min, and 100 μ l 1.0N salting liquids is added to terminate reaction.With
The light absorption value of microplate reader measurement wavelength 450nm.The curve that OD value corresponds to antibody extension rate is drawn, 1/2 " platform OD value " is found out
Corresponding antibody concentration A.Calculating affinity costant using following equation is 2.16L × mol-1。
Three, monoclonal antibody atopic and application effect
Immunogen solution is selected, the identification specificity of monoclonal antibody of the invention is detected with the method for immunoblotting, it is right
MSH2 albumen-KLH conjugate carries out 12.5% polyacrylamide gel electrophoresis.Then gel protein is turned with conventional wet robin
Move on to pvdf membrane.Film is placed in 5%BSA-TBST solution (albumen is face-down), 1h is closed in 37 DEG C of shaking tables, to eliminate non-spy
Anisotropic background.5%BSA-TBST is washed off with TBST after closing, the monoclonal antibody of MSH2 prepared by the present invention is added, in
Decolorization swinging table, which sways, is incubated for 1h.After washing film with TBST, secondary antibody is added, is incubated for 1h in decolorization swinging table, ties secondary antibody sufficiently with primary antibody
It closes.
Film is washed with TBST again, ECL colour reagent box is added, experimental result is as shown in Figure 2.MSH2 immunogene is in MSH2
Clear positive band, item are shown on (1B11-14-16) monoclonal antibody, HCT8 cell pyrolysis liquid and Biu-87 cell pyrolysis liquid
It is deeper with single and color, illustrate that the specific reaction of antibody and antigen is obvious.And the SP2-0 cell in the expression of MSH2 feminine gender is split
It solves on liquid without purpose band.
The dyeing of 6 organization chip of embodiment and identification
One, paraffin embedded tissues preparation process
HE slice dyeing is carried out to sample tissue, to determine lesion.It draws a circle to disease site, preparation punching.
When making acceptor wax block, plastics are placed on mold, the paraffin (fusing point is at 56~58 DEG C) of thawing are poured into mold, by tissue
Block adds appropriate wax liquor after being put into the wax liquor in mold be fully embedded in tissue block in wax liquor, by mould after being cooled to room temperature
Tool is put into -20 DEG C of refrigerator 6min, and wax stone is removed from the molds, and is sliced or is put into and saves backup in 4 DEG C of refrigerators.It is carried out after photo fix
Serial section, thickness are set to 4 μm, and serial section is floated in 40% alcohol, allows it to be unfolded naturally, then separated slice is transferred to
It opens up piece 30 seconds, is sliced with through the processed glass slide mount of 2%APES acetone solution, by manufactured organization chip in 45 DEG C of warm water
It is put into 60 DEG C of ovens and bakes piece 2 hours, take out room temperature cooling, be put into -4 DEG C of refrigerators and save.
Two, IHC dyeing and analysis
Conventional xylene dewaxes 3 times, and 6 minutes every time, aquation in 100%, 100%, 95%, 95%, 85% graded ethanol,
3 minutes every time, last tap water rinsed.Antigen retrieval is carried out, then slice is put into wet box, PBS is rinsed 3 × 3 minutes.Drop
Add 3%H2O2It is incubated for 10 minutes, PBS is rinsed 3 × 3 minutes.PBS is got rid of, peroxidase blocking reagent is added dropwise and is incubated at room temperature 10 points
Clock.Drying slice, is added dropwise the diluted primary antibody of proper proportion (diluting the dilution ratio according to antibody concentration come designerantibodies for the first time)
(25 DEG C) of room temperature are incubated for 1 hour, and PBS is rinsed 3 × 3 minutes, and secondary antibody is added dropwise and is incubated at room temperature 30 minutes, and PBS is rinsed 3 × 3 minutes, is got rid of
PBS is removed, is developed the color 3-10 minutes with the DAB developing solution of fresh configuration.Haematoxylin is redyed 20 seconds, and PBS returns indigo plant.According to 85% (3 points
Clock), 95% (3 minutes), 95% (3 minutes), 100% (3 minutes), 100% (3 minutes) alcohol gradient be successively dehydrated, finally
Transparent 10 minutes of dimethylbenzene twice, neutral gum mounting.
Immunohistochemical staining result is divided into: positive and negative.Positive expression must be in cell and the specific antigen portion of tissue
Position can just be considered as the positive.In tissue staining distribution clearly and in the accurate situation of cellular localization, coloration result is according to staining power
Difference carry out further division, it is specific as follows:
1, sample is weakly positive；Labeled as "+"；
2, sample is moderate positive；Labeled as " ++ "；
3, sample is High positive；Labeled as " +++ ".
4, sample is feminine gender, is labeled as "-".
Three, pattern detection result:
With anti-MSH2 protein monoclonal antibody (1B11-14-16) and the commercially available anti-MSH2 protein monoclonal antibody of control antibodies-
(mouse monoclonal antibody G219-1129) synchronizes inspection in 52 colorectal cancers, 34 prostate cancers, 29 bladder cancers, 47 gastric cancers
It surveys, as a result as shown in the table.
As the result is shown: clear and nothing is dyed in the dyeing accurate positioning of anti-MSH2 protein monoclonal antibody (1B11-14-16)
Unspecific staining, clean background illustrate anti-MSH2 protein monoclonal antibody (1B11-14-16) high specificity.And some cases
In, it is higher than using the positive cell number and positive strength of anti-MSH2 protein monoclonal antibody (1B11-14-16) using control examination
Agent illustrates that the sensibility of anti-MSH2 protein monoclonal antibody (1B11-14-16) and affinity are higher than commercial antibody.
And it is anti-with anti-MSH2 protein monoclonal antibody (1B11-14-16) and the commercially available anti-MSH2 protein monoclonal of control antibodies-
Body (mouse monoclonal antibody G219-1129), synchronizes detection on the organization chip for including 30 kinds of normal tissues, and sample is positive and negative
As a result consistent, illustrate that this antibody is suitable with commercial antibody in the specificity of normal tissue.
30 kinds of normal tissues include: brain, heart, cerebellum, oesophagus, adrenal gland, stomach, ovary, small intestine, pancreas, Colon and rectum,
Parathyroid gland, liver, hypophysis, salivary gland, testis, kidney, thyroid gland, prostate, mammary gland, uterus, spleen, bladder, tonsillotome, bone
Flesh, thymus gland (child), skin, marrow, peripheral nerve, lung, mesothelial cell.
Fig. 3 is that (MSH2 that a left side is 1B11-14-16, the right side are commercially available to Colorectal Carcinoma immunohistochemical staining result figure
MSH2).Wherein, the positive cell number and positive strength of the dyeing of the MSH2 of 1B11-14-16 are apparently higher than the dyeing of commercially available MSH2
Intensity illustrates that its susceptibility is higher.
Fig. 4 is Bladder Cancer immunohistochemical staining result figure (MSH2 that a left side is 1B11-14-16, the right side are commercially available MSH2).
Wherein, the positive strength of the dyeing of the MSH2 of 1B11-14-16 is apparently higher than the staining power of commercially available MSH2, illustrates its susceptibility
It is higher.
Fig. 5 is tonsil immunohistochemical staining result figure (MSH2 that a left side is 1B11-14-16, the right side are commercially available MSH2).
Wherein, the positive strength of the dyeing of the MSH2 of 1B11-14-16 is apparently higher than the staining power of commercially available MSH2, illustrates its susceptibility
It is higher.
Fig. 6 is appendix tissue immunohistochemical staining result figure (MSH2 that a left side is 1B11-14-16, the right side are commercially available MSH2).Its
In, the positive cell number of the dyeing of the MSH2 of 1B11-14-16 is apparently higher than the staining power of commercially available MSH2, illustrates its susceptibility
It is higher.
Sequence table
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tgcaaggcta ctggctacac attcactagg tactggatag agtgggtaaa agagaggcct 180
ggacatggcc ttgagtggat tggagagatt ttacctggaa gtggtggtac taactacaat 240
gagaaattca agggcaaggc cacattcact gcagatacat cctccaacac agtccacatg 300
caactcaaca gcctgacatc ggaagactct gccgtctatt actgtgtaaa gagagagggt 360
ccctactggg gccgagggac tctggttact gtctctgca 399
<210> 5
<211> 393
<212> DNA
<213>artificial sequence (Artificial)
<400> 5
atgaagttgc ctgttaggct gttggtgctg atgttctgga ttcctgtttc ctccagtgat 60
gttttgatga cccaaactcc actctccctg cctgtcagtc ttggagatca agcctccatc 120
tcttgcagat ctagtcagag tattgtatat aggactggaa gcaccttttt agaatggtac 180
ctgcagaaac caggccagtc tccaaagctc ctgatctaca aagtttccaa ccgattttct 240
ggggtcccag acaggttcag tggcagtgga tcagggacag atttcacact caagatcagc 300
agagtggagg ctgaggatct gggagtttat tactgctttc aaggttcaca tcttccgtac 360
acgttcgggg gggggaccaa gctggaaata aaa 393
Claims (10)
1. a kind of anti-MSH2 protein monoclonal antibody, which is characterized in that the ammonia of the monoclonal antibody heavy and light chain variable region
Base acid sequence is amino acid sequence shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.
2. monoclonal antibody according to claim 1, which is characterized in that the monoclonal antibody heavy and light chain variable region
Amino acid sequence is coded by nucleotide sequence shown in SEQ ID NO.4 and SEQ ID NO.5 respectively.
3. monoclonal antibody according to claim 1, which is characterized in that the monoclonal antibody specificity identifies MSH2 egg
It is white.
4. monoclonal antibody according to claim 3, which is characterized in that the monoclonal antibody specificity identifies MSH2 egg
Amino acid sequence shown in white middle SEQ ID NO.1.
5. a kind of anti-MSH2 protein monoclonal antibody is generated by the hybridoma cell line that deposit number is CGMCC NO.17405.
6. monoclonal antibody according to claim 1, which is characterized in that the anti-MSH2 albumen is mouse IgG2bHypotype list
Clonal antibody.
7. a kind of preparation method of anti-MSH2 protein monoclonal antibody, which is characterized in that choose MSH2 protein amino acid sequence the
919, to the 934th amino acids, are added sulfydryl modification in its N-terminal and carry out coupling as immunogene with carrier protein KLH.
8. the hybridoma cell line of one plant of anti-MSH2 protein monoclonal antibody of secretion, the cell line is mouse hybridoma cell system
1B11-14-16, the cell line are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number
Are as follows: CGMCC NO.17405.
9. any anti-MSH2 protein monoclonal antibody of claim 1-6, the purposes in the detection of MSH2 protein immunization.
10. immune detection according to claim 9, which is characterized in that the immune detection includes immunohistochemical method,
Western blot and enzyme-linked immunization.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113845595A (en) * | 2021-11-16 | 2021-12-28 | 福州迈新生物技术开发有限公司 | anti-GH protein monoclonal antibody, cell line, preparation method and application thereof |
| CN113845593A (en) * | 2021-10-27 | 2021-12-28 | 福州迈新生物技术开发有限公司 | anti-alpha-SMA protein monoclonal antibody, cell line and application thereof |
| CN117487007A (en) * | 2023-09-26 | 2024-02-02 | 武汉爱博泰克生物科技有限公司 | Monoclonal antibody combined with human MutS homologous protein 2 and application thereof |
| CN117487007B (en) * | 2023-09-26 | 2024-05-10 | 武汉爱博泰克生物科技有限公司 | Monoclonal antibody combined with human MutS homologous protein 2 and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5907079A (en) * | 1996-01-18 | 1999-05-25 | Amgen Canada Inc. | MSH2 disrupted mice develop lymphomas |
| US20120004136A1 (en) * | 2009-03-19 | 2012-01-05 | Institut Gustave-Roussy | Msh2 and adjuvant cisplatin-based chemotherapy in non-small-cell lung cancer |
| CN108623683A (en) * | 2018-05-30 | 2018-10-09 | 福州迈新生物技术开发有限公司 | The monoclonal antibody and its cell strain, preparation method and application of anti-Pax-5 albumen |
-
2019
- 2019-06-19 CN CN201910529447.0A patent/CN110218251B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5907079A (en) * | 1996-01-18 | 1999-05-25 | Amgen Canada Inc. | MSH2 disrupted mice develop lymphomas |
| US20120004136A1 (en) * | 2009-03-19 | 2012-01-05 | Institut Gustave-Roussy | Msh2 and adjuvant cisplatin-based chemotherapy in non-small-cell lung cancer |
| CN108623683A (en) * | 2018-05-30 | 2018-10-09 | 福州迈新生物技术开发有限公司 | The monoclonal antibody and its cell strain, preparation method and application of anti-Pax-5 albumen |
Non-Patent Citations (3)
| Title |
|---|
| FREDRICK S. LEACH等: "Expression of the Human Mismatch Repair Gene hMSH2 in Normal and Neoplastic Tissues", 《CANCER RESEARCH》 * |
| UNIPROT: "P43246 MSH2_Human", 《HTTPS://WWW.UNIPROT.ORG/UNIPROTKB/P43246/ENTRY》 * |
| 彭芳等: "结直肠癌错配修复蛋白MLH1,PMS2,MSH2和MSH6及P16的表达及意义", 《实验与检验医学》 * |
Cited By (7)
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|---|---|---|---|---|
| CN113845593A (en) * | 2021-10-27 | 2021-12-28 | 福州迈新生物技术开发有限公司 | anti-alpha-SMA protein monoclonal antibody, cell line and application thereof |
| CN113845593B (en) * | 2021-10-27 | 2023-03-10 | 福州迈新生物技术开发有限公司 | anti-alpha-SMA protein monoclonal antibody, cell line and application thereof |
| CN113845595A (en) * | 2021-11-16 | 2021-12-28 | 福州迈新生物技术开发有限公司 | anti-GH protein monoclonal antibody, cell line, preparation method and application thereof |
| CN113845595B (en) * | 2021-11-16 | 2023-03-10 | 福州迈新生物技术开发有限公司 | anti-GH protein monoclonal antibody, cell line, preparation method and application thereof |
| CN117487007A (en) * | 2023-09-26 | 2024-02-02 | 武汉爱博泰克生物科技有限公司 | Monoclonal antibody combined with human MutS homologous protein 2 and application thereof |
| CN117487007B (en) * | 2023-09-26 | 2024-05-10 | 武汉爱博泰克生物科技有限公司 | Monoclonal antibody combined with human MutS homologous protein 2 and application thereof |
| CN117467003B (en) * | 2023-10-07 | 2024-05-17 | 武汉爱博泰克生物科技有限公司 | Anti-human MSH6 protein rabbit monoclonal antibody and application thereof |
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| CN110218251B (en) | 2022-10-25 |
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