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CN109576229A - A kind of monoclonal antibody hybridoma cell strain and its application for secreting anti-AGR2 - Google Patents

A kind of monoclonal antibody hybridoma cell strain and its application for secreting anti-AGR2 Download PDF

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CN109576229A
CN109576229A CN201811522600.9A CN201811522600A CN109576229A CN 109576229 A CN109576229 A CN 109576229A CN 201811522600 A CN201811522600 A CN 201811522600A CN 109576229 A CN109576229 A CN 109576229A
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agr2
cell line
hybridoma cell
monoclonal antibody
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王征
王琳
胡嘉
储亚楠
田少博
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Union Hospital Tongji Medical College Huazhong University of Science and Technology
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Abstract

本发明涉及一种针对AGR2的单克隆抗体杂交瘤细胞株及其应用，所述杂交瘤细胞株的分类命名为：小鼠杂交瘤细胞株，该细胞株于2018年08月28日保藏于中国普通微生物菌种保藏管理中心。保藏中心登记入册保藏编号为CGMCC No.16288。由所述杂交瘤细胞株分泌得到的单克隆抗体能够有效检测结肠癌细胞如HT29细胞及结肠癌组织中的AGR2，特异性好。此外还可阻断AGR2促进结肠癌的转移，具体为体外实验抑制结肠癌细胞的迁移。

The present invention relates to a monoclonal antibody hybridoma cell line against AGR2 and an application thereof. The classification name of the hybridoma cell line is: mouse hybridoma cell line, and the cell line was preserved in China on August 28, 2018 Ordinary Microbial Culture Collection and Management Center. The deposit number registered in the deposit center is CGMCC No.16288. The monoclonal antibody secreted by the hybridoma cell line can effectively detect AGR2 in colon cancer cells such as HT29 cells and colon cancer tissue, with good specificity. In addition, AGR2 can also be blocked to promote colon cancer metastasis, specifically inhibiting colon cancer cell migration in vitro.

Description

A kind of monoclonal antibody hybridoma cell strain and its application for secreting anti-AGR2
Technical field
The invention belongs to monoclonal antibody art, be related to a kind of stable expression can specificity for AGR2 (preceding gradient albumen 2) application of the cell strain of monoclonal antibody and the cell strain and the monoclonal antibody of generation.
Background technique
AGR2 is gradient albumen -2(XAG-2 before Africa xenopus (Xenopus)) homologous protein in the mankind.In human body AGR2 albumen is mainly related with the secreting function of body of gland, therefore in some histoorgans rich in mucus or with secreting function Middle high expression, such as Colon and rectum, stomach, mammary gland and prostate.AGR2 albumen belongs in PDI family (protein disulfide isomerase) A member, be primarily targeted in endoplasmic reticulum, while can also be secreted extracellularly.In the humoral specimens such as serum, urine It can detect the presence of secreting type AGR2 albumen.People find earliest AGR2 it is related to tumour be in breast cancer, with research Deepen continuously, it has been found that the occurrence and development that AGR2 promotees cancer molecule and kinds of tumors as one have close ties, AGR2 Effect in tumour is increasingly valued by people.
Inventor has found that exogenous AGR2 can promote the migration of colorectal cancer cell in early-stage study, expression quantity with Tumor prognosis is closely related.Furthermore studies have found that AGR2 can interact with extracellular matrix promotes tumor development. Therefore AGR2 is likely to become a potential cancer target.It is therefore desirable to develop the Dan Ke for detecting, targeting AGR2 Grand antibody provides new thinking and tool for diagnosis, the treatment of cancer, has great importance and application value.
This patent uses the AGR2 full-length proteins with his label that ICR mouse is immunized, because of mark entrained by immune protein Label molecular weight is smaller, and immunogenicity is lower for the macromoleculars label such as MBP, GST, and the antibody specificity of generation is preferable. Furthermore the ICR mouse correlation fusion stability used is high, and rejection is small, and the Antibody stability that the later period generates is preferable.
Summary of the invention
One of the objects of the present invention is to provide a kind of hybridoma cell strains of monoclonal antibody for secreting anti-AGR2 albumen. The classification naming of the hybridoma cell strain are as follows: mouse hybridoma cell strain, the cell strain were preserved on 08 28th, 2018 China Committee for Culture Collection of Microorganisms's common micro-organisms center, depositary institution address: BeiJing, China.Collection registration Entering volume number is CGMCC No.16288.
The second purpose of the present invention is to provide a kind of monoclonal antibodies that can specifically identify, target AGR2 albumen 10G11.The antibody is secreted by above-mentioned hybridoma cell strain.
The three of the object of the invention are the provision of the above-mentioned application for AGR2 hybridoma or monoclonal antibody.
Above-mentioned hybridoma cell strain is preparing AGR2 Protein Detection or is diagnosing the application in related drugs and reagent；
Application of the above-mentioned hybridoma cell strain in the drug and preparation of preparation treating cancer；
Said monoclonal antibody is preparing AGR2 Protein Detection or is diagnosing the application in related drugs and reagent；
Application of the said monoclonal antibody in the drug and preparation of preparation treating cancer.
The present invention provides a kind of methods for preparing AGR2 monoclonal antibody, mainly comprise the steps that
1) building of recombinant expression carrier
According to AGR2 open reading frame sequence, design primer expands the code area AGR2, is inserted by EcoRI and XhoI restriction enzyme site Into pET28a carrier, pET28a-AGR2-His recombinant expression carrier is constructed.
) AGR2 recombinant protein expression and purifying
The recombinant expression carrier built in step 1) is converted into BL21 competent bacteria, the expression of IPTG inducible protein surpasses Sound cracks bacterium, and centrifugation obtains albumen supernatant.By affinity chromatography column purification, the AGR2 albumen of purifying is obtained.
) monoclonal antibody screening and preparation
Take the spleen of mouse thin after initial immunity and booster immunization the AGR2 protein immunization ICR female mice purified in step 2) Born of the same parents and myeloma cell SP2/0 fusion, limiting dilution assay obtain monoclonal.It is thin that positive hybridoma is filtered out by ELISA method Born of the same parents' strain, obtains the hybridoma cell strain that can secrete the monoclonal antibody for AGR2 albumen, is named as 10G11, subtype identification For IgG1 type.Hybridoma cell strain is seeded to mouse peritoneal, mouse ascites is collected, passes through Protein A/G antibody purification.
The present invention has the advantages that
The antibody that inventor is prepared by the above method can effectively detect colon cancer cell such as HT29 and colon cancer tissue In AGR2, specificity it is good.AGR2 can be additionally blocked to promote the transfer of colon cancer, specially experiment in vitro inhibits colon cancer thin The migration of born of the same parents.The colon cancer cell includes SW48 and HT29, the external shift experiment experimental method common using this field It determines, as (Transwell experiment) is tested in the tumor migration cell in attached drawing 2.
Detailed description of the invention
Fig. 1 shows the design drawing in pET28a-AGR2-C-His plasmid cloning site, and wherein dash area is the coding of AGR2 Area；
Fig. 2 shows expression of results of the AGR2 albumen in supernatant of bacteria solution and precipitating；
Fig. 3 shows AGR2 protein expression and purification result；
Fig. 4 shows the result of AGR2 antibody specificity identification AGR2 albumen in Western Blot experiment screening mice serum；
Fig. 5 shows the result of AGR2 antibody blocking colon cancer cell migration in Transwell experiment screening mice serum；
Fig. 6 shows that AGR2 antibody specificity identifies colon cancer in Western Blot experiment screening monoclonal cell strain cell conditioned medium The result of endogenous AGR2 albumen in cell；
Fig. 7 shows the knot of AGR2 antibody blocking colon cancer cell migration in Transwell testing sieve clonal cell line cell conditioned medium Fruit；
The AGR2 antibody purity testing result of Fig. 8 display purifying；
Fig. 9 shows that Transwell experimental verification AGR2 monoclonal antibody can block exogenous AGR2 albumen to promote SW48 cell Migration；
Figure 10 shows that Transwell experiment detection AGR2 monoclonal antibody can block secreting type AGR2 to promote moving for HT29 cell It moves；
Figure 11 shows endogenous in Western Blot experiment detection AGR2 monoclonal antibody specificity identification colon cancer cell The result of AGR2 albumen；
Figure 12 shows endogenous AGR2 egg in immunofluorescence experiment detection AGR2 monoclonal antibody specificity identification colon cancer cell White result.
Specific embodiment
The specific embodiment of description is said to specifications, and those skilled in the art can reproduce its implementation according to its description Example simultaneously can prepare its product.
All terms in specification are all made of technical term well known in the art (english abbreviation including raw material etc.), nothing Method is using technical term, and specification can explain and illustrate to term used, so that those skilled in the art understand that it contains Justice.
Unless specifically indicated, technological means employed in specification is the ordinary skill in the art, is delivering text It can be consulted in offering.
The building of 1 AGR2 recombinant expression plasmid of embodiment
AGR2 open reading frame (ORF) nucleotide sequence is obtained from the website NCBI.Using the genome cDNA of people as template, respectively with AGR2-F, AGR2-R are primer amplification AGR2 overall length, recycle target fragment, carry out merging weight with the pET28a carrier after digestion Group.The gene coding region AGR2 is inserted by the cloning site design drawing of carrier as shown in Figure 1, by restriction enzyme site EcoRI and XhoI Into pET28a carrier, ET28a-AGR2-His recombinant expression carrier is constructed.
Step 1) target gene design of primers
Design synthesizes following primer, for expanding AGR2 open reading frame overall length:
AGR2-F:ATGGGTCGCGGATCCGAATTCATGGAGAAAATTCCAGTGTC；
AGR2-R:GTGGTGGTGGTGGTGCTCGAGTCAATTCAGTCTTCAGCAACT.
The amplification of step 2 target gene
Using human genome cDNA as template, respectively using AGR2-F, AGR2-R as primer amplification AGR2 overall length, amplification system is as follows:
Amplification condition is as follows: 95 DEG C of initial denaturation 3min；95 DEG C of denaturation 15s, 56 DEG C of annealing 15s, 72 DEG C of extension 30s, totally 30 are followed Ring；Last 72 DEG C re-extend 5min；
Step 3) digestion carrier
With EcoRI and XhoI double digestion pET28a carrier, 37 DEG C digestion 1 hour.Digestion system is as follows:
Step 4) carrier is connect with target gene
It carries out merging recombination with the target gene of amplification after carrier after digestion is carried out glue recycling, 37 DEG C are reacted 30 minutes.Recombination System is as follows:
Step 5) plasmid identification
Connection product in above-mentioned steps is converted to DH5 α competent bacteria: taking 50ul competent bacteria from -80 DEG C of refrigerators, on ice After thawing, above-mentioned connection product is added, after ice sets 20min, 42 DEG C of water-bath heat shock 90s, continue ice set be added after 10min 1ml without Resistance LB culture medium, 5000rpm centrifugation 3min collects appropriate bacterium solution after 37 degree of shaking tables are incubated for 45min, be coated on containing card that In the LB plate of mycin antibiotic, flat-plate inverted is placed in 37 DEG C of incubators and is incubated overnight；Picking monoclonal colonies are containing card It is cultivated in the fresh culture of that mycin, extracts and plasmid is stored in -20 DEG C after plasmid order-checking is identified correctly.
The expression and purification of 2 AGR2 recombinant protein of embodiment
Step 1) expresses albumen and identification in a small amount
The pET28a-AGR2-His plasmid for taking 100ng to build is converted into the BL21 competent E.coli of 50ul；
Ice sets 42 DEG C of heat shock 90s after 20min；Continue the LB culture medium that ice sets addition non-resistant after 10min；37 degree of shaking tables are incubated for 5000rpm is centrifuged 3min and collects appropriate bacterium solution after 45min, is coated in the LB plate of antibiotic containing kanamycin, by plate Inversion is put in 37 DEG C of incubators and is incubated overnight；
Picking monoclonal colonies, which are cultivated in 2ml fresh culture containing kanamycin and save appropriate bacterium solution and stay, does bacterium Kind, 37 DEG C, 200rpm cultivates the IPTG that final concentration of 500uM is added when reaching 0.5 to OD value in 30 DEG C of continuation inducing expression 6h；
8000rpm is centrifuged 2min and collects thallus；
Thallus is resuspended with 150ul PBS, in 4 DEG C after the bacterium of ultrasound cracking on ice, 8000rpm is centrifuged 15min, collects supernatant egg It is white；Albumen is extracted into precipitating RIPA lysate dissolution；
5min is boiled for 95 DEG C after taking 50ul supernatant protein and protein precipitation to add Loading Buffer, is reflected by SDS-PAGE gel electrophoresis It is fixed, coomassie brilliant blue staining.
As a result as shown in Fig. 2, AGR2 albumen great expression is in supernatant precipitating, the supernatant after subsequently selected ultrasound is used for Large-scale purification AGR2 albumen.
Step 2 great expression albumen
It takes the conversion of -80 DEG C of preservations to have the BL21 strain of pET28a-AGR2-His plasmid, is inoculated into containing kalamycin resistance In LB culture, 37 DEG C of shaking table cultures are activated 16 hours；
The bacterium solution of activation is transferred in the 400ml LB culture medium containing kanamycins, 37 DEG C of cultures add to OD value about 0.5 or so Enter 500uM IPTG 30 DEG C inducing expression 6 hours；
8000rpm is centrifuged 2min and collects thallus, and appropriate PBS(is added containing protease inhibitors after weighing bacterium precipitating quality), on ice Ultrasound cracking bacterium；
4 DEG C, 8000rpm is centrifuged 15min, collects supernatant solution.
Step 3) protein purification
Affinity chromatography column method purifying protein is used in the present invention.
Assemble chromatographic column, after nickel column stratification, with 10 times of column volume Binding Buffer (10mM imidazoles, 20mM Tris-HCl, 500mM NaCl) it cleans and balances nickel column, it has been careful not to bubble；
After the supernatant protein gathered the filter filtering of 0.45um, mixes and be loaded in equal volume with Binding Buffer In nickel column, collection flows through liquid, upper prop is repeated twice, to ensure that albumen is sufficiently integrated to nickel column；
Nickel column is balanced again with 10 times of column volume Binding Buffer, washes away unbonded foreign protein；
Destination protein is eluted with Elution Buffer (250mM imidazoles, 20mM Tris-HCl, 500mM NaCl), is collected respectively The identification of SDS-PAGE gel electrophoresis is carried out afterwards.
As a result as shown in figure 3, result prompts most of albumen to elute success, the protein liquid of elution is then placed into dialysis Bag (specification 3500) is dialysed.Dialyzate imidazole concentration since 200mM gradually dialyse by near 30mM, every time 0.8 times of dilution Albumen is collected after 48 hours, measurement concentration is 400ng/ul, and -80 DEG C of preservations are placed in albumen packing.
The screening and preparation of 3 AGR2 monoclonal antibody of embodiment
By the AGR2 albumen of purifying with 60ug/ dosage only to 4 SPF grade ICR female mices progress initial immunities, after two weeks with 30ug/ dosage carries out booster immunization, and after four booster immunizations, eye socket takes blood, with ELISA method detection mice serum effect Valence (table 1) and Western Blot detection serum specificity (Fig. 4), and blocked with Transwell method measurement mice serum The rush migration effect (Fig. 5) of AGR2.Selection serum titer highest simultaneously has the mouse for blocking AGR2 to promote migration progress cell to melt Close experiment；Mouse boosting cell suspension and SP2/0 cell in good condition are taken, is merged with PEG method.Cell is containing after fusion Screening and culturing is carried out in the semisolid culturemedium of HAT.
1 mice serum potency of table
Extension rate 200 400 800 1600 3200 6400 12800 25600 51200 102400 Blank It is negative
AGR2-1 1.104 0.971 0.815 0.604 0.431 0.273 0.154 0.092 0.078 0.062 0.019 0.087
AGR2-2 1.069 1.078 0.99 0.835 0.671 0.505 0.338 0.166 0.136 0.093 0.021 0.083
AGR2-3 1.292 1.294 1.234 1.177 1.101 0.927 0.688 0.458 0.346 0.261 0.02 0.076
AGR2-4 0.238 0.34 0.334 0.228 0.149 0.102 0.072 0.048 0.059 0.044 0.044 0.078
Note: potency is the corresponding dilution of the minimum OD reading greater than maximum OD/2.
Step 1) animal immune
(1) with the AGR2 albumen of purifying, by 60ug albumen/mouse amount, 4 SPFICR female mices of subcutaneous initial immunity, Number are as follows: 1#, 2#, 3#, 4#；
(2) subcutaneous booster immunization is carried out after two weeks, and the amount of being immunized is 30ug albumen/only, altogether booster immunization 4 times.
Step 2 ELISA method measures sero-immunity potency
(1) eye socket takes blood；
(2) with the AGR2 albumen of purifying, 2ug/ml, 4 °C of coatings are overnight；
(3) 2% milk, 37 °C of closing 2h；
(4) it is washed 3 times with washing lotion, mice serum, mice serum 2 times of gradient dilutions, blank control since 200 times is added It (blank) is PBS, negative control (negative) is 200 times of negative serum dilutions；
(5) it is washed 3 times with washing lotion.The secondary antibody that PBS dilutes 20000 times, the hole 100ul/, 37 DEG C of incubators, 1h is added；
(6) it is washed 3 times after taking out with washing lotion；
(7) it develops the color, the hole developing solution 100ul/, developing time is 5min or so.Every hole is added 50ul terminate liquid and terminates；
(8) dual wavelength (450,630) surveys light absorption value, and record saves data.
Step 3) Western Blot screens mice serum
(1) PBS that cell is pre-chilled is washed twice, RIPA lysate (containing protease inhibitors) is added, pressure-vaccum mixes；
(2) ice sets 15min after the 15s of ultrasound cracking on ice；12000rpm；
Supernatant is transferred to after (3) 4 DEG C of centrifugation 15min and newly manages and marks；
(4) protein quantification is carried out according to BCA protein quantification kit, be added after albumen sample-loading buffer in each sample in 98 DEG C Metal bath 5min；
(5) after 12% PAGE glue electrophoresis, 180mA Constant Electric Current turns 100min；
(6) 1h is closed with 5% milk room temperature of TBST configuration；
(7) 4 DEG C of primary antibody overnight incubations.
(8) TBST is washed film 3 times, after each 5min, is incubated at room temperature secondary antibody 1h, TBST washes film post-exposure.
The measurement of step 4) serum barrier effect
(1) colon cancer cell SW48 digestion counts and after suitable cell suspension is made, by the cell Transwell be put into 24 holes without In bacterium culture plate；
(2) 500ul fresh complete medium is added in cell lower layer, and the AGR2 egg of 100ng/ml is added in experimental group lower layer culture medium White and appropriate mice serum；
(3) cell suspension 200ul is added in cell upper layer, and cell quantity 50,000 is added；
(4) it after cultivating 12 hours in the incubator, takes out cell and fixes 15 minutes with 4% paraformaldehyde room temperature, then crystal violet Dyeing 20 minutes.The cell that cell upper layer is not passed through gently is wiped with cotton swab, with micro- sem observation and is photographed to record.
The results are shown in Table 1, and AGR2 albumen is after initial immunity and three times booster immunization mouse, the serum of 3# mouse ELISA potency highest；Fig. 4 shows Western Blot testing result, and the serum of 3# mouse can detect AGR2 in HT29 cell Expression, and express less in SW48 cell, this is consistent with document report；Fig. 5 shows Transwell experimental result, 3# mouse Serum can block exogenous AGR2 to the rush migration of SW48 cell.Therefore next we select 3# mouse to carry out cell Fusion experiment before cell fusion, then carries out abdominal cavity impact and is immunized once.
The experiment of step 5) cell fusion
Selection serum titer highest simultaneously has the mouse for blocking AGR2 to promote migration, with immunogene 50ugAGR2 albumen, abdominal cavity punching The mouse is hit, cell fusion experiment is ready for.
(1) it is blown and beaten from culture bottle wall by SP2/0 cell in good condition is soft, is drawn into 50ml centrifuge tube In；
(2) mouse plucks eyeball and takes blood, then neck is drawn to put to death, is put into 75% alcohol and impregnates 5min；
(3) IMDM that a small amount of serum-free is poured into plate, cell sieve and plunger are put into plate.With scissors and tweezer Son removes the spleen of mouse, is put into cell sieve.Lightly spleen is sufficiently pulverized with the inner core of syringe, the cell ground is inhaled Enter into the centrifuge tube of dress SP2/0, is centrifuged 1500rad/min, 5min；
(4) thymus gland that mouse is removed with scissors and tweezers, pulverizes.By the thymocyte ground into 15ml centrifuge tube, add The HT of the HAT of 2ml, 2ml are placed on spare in incubator；
(5) cell that will be centrifuged, outwells supernatant, and cell is carefully gently blown to even, centrifugation with the IMDM of serum-free (1500rad/min, 5min)；
(6) cell conditioned medium being centrifuged is outwelled as far as possible.The abundant suspension cell in centrifuge tube bottom is patted, centrifuge tube is put into 37 DEG C of temperature In water, it is slowly added to the PEG of 1ml in 1 minute, after adding, 1min is stood in warm water.Then 2ml is slowly added in 2min Serum-free IMDM, the IMDM of 8ml serum-free is then slowly added in 2min.It is centrifuged (1000rad/min, 5min)；
(7) supernatant is outwelled, the serum of 10ml is added, careful blow cell is even, pours into the ready thymocyte in front.Again plus Enter the sterilized semisolid culturemedium of 25ml, mixes well.Then it uniformly pours into 30 Tissue Culture Dish.By Tissue Culture Dish It is put into wet box, is then placed in incubator and cultivates.
Step 6) monoclonal cell primary dcreening operation
(1) after fused cell culture 10 days, start kind of plate and select monoclonal.The hybridoma pancreatin of routine culture is disappeared For kind in 10 96 orifice plates, dilution standard is that every hole only has 1 cell after changing and counting dilution；
(2) after cultivating 5 days, ELISA method is used to the clone selected, carries out first time screening.ELISA laboratory operating procedures with Step 2 is similar in this example, the difference is that primary antibody used in this step is monoclonal cell culture supernatant.
As a result as shown in table 2- table 11,24 plants of positive monoclonal cell strains is filtered out altogether from 10 96 orifice plates and are compiled Labelled notation.
2 fused cell screen of table selects (plate 1)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.036 0.014 0.025 0.021 0.018 0 0.041 0.013 0.034 0.001 0.001 0.021
B 0.026 0.001 0.001 0.001 0.001 0 0.001 0.001 0.001 0.001 0.001 0.028
C 0.017 0.019 0.013 0.01 0 0.016 0.014 0.001 0.007 0.001 0.007 0.001
D 0.023 0.017 0.02 0.013 0.012 0.339 0.012 0.001 0.001 0.011 0 0.015
E 0.015 0.014 0.017 0.014 0.022 0.027 0.022 0.01 0.031 0.007 0.012 0.011
F 0.014 0.008 0.018 0.001 0.001 0.001 0.008 0.01 0.013 0.01 0.001 0.031*
G 0.025 0.019 0.017 0.01 0.022 0.014 0.015 0.015 0.015 0.03 0.014 0.011*
H 0.017 0.195 0.038 0.052 0.066 0.114 0.041 0.047 0.011 0.035 0.034 0.659*
Note: add *'s to be followed successively by negative control, blank control and positive control from top to bottom；Overstriking font is positive gram of screening It is grand
3 fused cell screen of table selects (plate 2)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.063 0.03 0.026 0.036 0.034 0.029 0.041 0.047 0.053 0.034 0.03 0.046
B 0.026 0.024 0.018 0.026 0.025 0.025 0.035 0.036 0.644 0.026 0.024 0.028
C 0.028 0.019 0.032 0.011 0.001 0.015 0.024 0.023 0.018 0.016 0.029 0.042
D 0.022 0.016 0.021 0.024 0.022 0.019 0.023 0.021 0.031 0.038 0.018 0.03
E 0.014 0.014 0.017 0.014 0.022 0.027 0.021 0.01 0.021 0.018 0.025 0.033
F 0.014 0.008 0.001 0.001 0.001 0 0.009 0.009 0.013 0.011 0.001 0.031*
G 0.015 0.02 0.017 0.01 0.022 0.014 0.015 0.015 0.016 0.03 0.037 0.023*
H 0.036 0.024 0.038 0.063 0.038 0.029 0.111 0.104 0.06 0.035 0.05 0.792*
Note: with table 2
4 fused cell screen of table selects (plate 3)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.036 0.032 0.025 0.018 0.019 0.017 0.026 0.013 0.028 0.019 0.019 0.024
B 0.024 0.001 0.001 0.001 0.012 0.001 0.001 0.001 0.029 0.001 0.001 0.001
C 0.017 0.018 0.014 0.01 0.014 0.012 0.014 0.008 0.024 0.009 0.018 0.016
D 0.024 0.016 0.02 0.013 0.012 0.018 0.022 0.012 0.139 0.011 0.016 0.016
E 0.025 0.014 0.014 0.013 0.011 0.015 0.022 0.011 0.022 0.016 0.023 0.029
F 0.033 0.025 0.026 0.021 0.024 0.024 0.018 0.009 0.014 0.023 0.027 0.042*
G 0.026 0.02 0.017 0.01 0.026 0.014 0.015 0.016 0.015 0.023 0.02 0.029*
H 0.06 0.041 0.024 0.026 0.017 0.044 0.028 0.013 0.025 0.067 0.043 0.815*
Note: with table 2
5 fused cell screen of table selects (plate 4)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.036 0.014 0.025 0.008 0.001 0.001 0.01 0.013 0.001 0.001 0.001 0.001
B 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
C 0.017 0.018 0.014 0.011 0.001 0.001 0.014 0.001 0.007 0.14 0.007 0.016
D 0.023 0.017 0.02 0.013 0.011 0.018 0.013 0.001 0.001 0.011 0.001 0.001
E 0.015 0.014 0.045 0.014 0.012 0.015 0.022 0.011 0.011 0.016 0.012 0.01
F 0.033 0.026 0.735 0.021 0.014 0.024 0.018 0.01 0.014 0.01 0.014 0.042*
G 0.025 0.019 0.017 0.01 0.011 0.014 0.015 0.015 0.015 0.008 0.029 0.01*
H 0.016 0.013 0.024 0.013 0.001 0.084 0.054 0.013 0.108 0.016 0.014 0.66*
Note: with table 2
6 fused cell screen of table selects (plate 5)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.034 0.03 0.015 0.017 0.001 0.013 0.02 0.012 0.017 0.009 0.03 0.033
B 0.008 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001 0.001
C 0.01 0.009 0.007 0.01 0.001 0.015 0.011 0.01 0.018 0.001 0.012 0.02
D 0.055 0.017 0.008 0.001 0.001 0.01 0.01 0.007 0.009 0.001 0.019 0.029
E 0.015 0.014 0.016 0.014 0.001 0.012 0.021 0.01 0.022 0.018 0.025 0.506
F 0.014 0.043 0.001 0.001 0.001 0.001 0.008 0.009 0.012 0.011 0.001 0.017*
G 0.015 0.02 0.016 0.01 0.001 0.014 0.015 0.015 0.016 0.011 0.015 0.023*
H 0.022 0.024 0.026 0.024 0.001 0.03 0.019 0.028 0.047 0.017 0.036 0.653*
Note: with table 2
7 fused cell screen of table selects (plate 6)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.051 0.03 0.025 0.017 0.039 0.025 0.02 0.027 0.036 0.034 0.029 0.056
B 0.019 0.001 0.001 0 0.001 0.016 0.015 0.016 0.021 0.018 0.017 0.036
C 0.041 0.009 0.001 0.011 0.015 0.015 0.023 0.02 0.018 0.019 0.012 0.021
D 0.219 0.016 0.007 0.001 0.016 0.519 0.024 0.02 0.019 0.016 0.019 0.029
E 0.326 0.014 0.016 0.014 0.02 0.032 0.02 0.026 0.022 0.017 0.024 0.038
F 0.051 0.066 0.025 0.019 0.027 0.033 0.036 0.032 0.054 0.035 0.037 0.065*
G 0.206 0.564 0.097 0.011 0.018 0.014 0.015 0.015 0.016 0.028 0.033 0.023*
H 0.034 0.269 0.062 0.034 0.034 0.03 0.038 0.04 0.036 0.057 0.036 1.007*
Note: with table 2
8 fused cell screen of table selects (plate 7)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.035 0.031 0.33 0.016 0.025 0.025 0.029 0.016 0.015 0.001 0.016 0.024
B 0.018 0.001 0.001 0 0.001 0.016 0.001 0.001 0.001 0.019 0.001 0.001
C 0.014 0.009 0.001 0.012 0.001 0.014 0.024 0.001 0.018 0.001 0.012 0.185
D 0.021 0.016 0.007 0.001 0.001 0.02 0.013 0.011 0.01 0.016 0.008 0.009
E 0.027 0.014 0.016 0.013 0.009 0.022 0.02 0.012 0.022 0.017 0.024 0.018
F 0.019 0.016 0.015 0.019 0 0.012 0.014 0.001 0.01 0.013 0.021 0.035*
G 0.023 0.014 0.012 0.011 0.008 0.014 0.015 0.014 0.016 0.01 0.304 0.022*
H 0.034 0.047 0.016 0.034 0.017 0.017 0.038 0.014 0.017 0.014 0.015 0.779*
Note: with table 2
9 fused cell screen of table selects (plate 8)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.035 0.03 0.022 0.016 0.012 0.025 0.029 0.034 0.014 0.017 0.015 0.024
B 0.008 0.007 0 0 0 0.016 0.001 0.001 0.001 0.116 0.001 0.019
C 0.014 0.009 0.001 0.012 0 0.014 0.01 0.001 0.018 0.001 0.011 0.023
D 0.046 0.026 0.017 0 0.001 0.289 0.013 0.011 0.011 0.016 0.009 0.02
E 0.027 0.014 0.312 0.013 0.009 0.022 0.02 0.012 0.011 0.017 0.023 0.018
F 0.031 0.017 0.015 0.001 0 0.011 0.014 0.001 0.011 0.013 0.021 0.035*
G 0.023 0.014 0.012 0.011 0.008 0.014 0.014 0.015 0.016 0.01 0.027 0.022*
H 0.022 0.022 0.027 0.017 0.029 0.017 0.021 0.026 0.017 0.048 0.028 0.597*
Note: with table 2
10 fused cell screen of table selects (plate 9)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.035 0.03 0.022 0.463 0.012 0.319 0.029 0.012 0.015 0.008 0.015 0.024
B 0.008 0.007 0.001 0.001 0.001 0.016 0.001 0.001 0.001 0.001 0.001 0.001
C 0.014 0.009 0.018 0.023 0 0.014 0.011 0.001 0.018 0.001 0.011 0.022
D 0.015 0.077 0.018 0.001 0.001 0.037 0.013 0.011 0.011 0.091 0.008 0.02
E 0.027 0.014 0.136 0.013 0.009 0.022 0.031 0.011 0.011 0.017 0.023 0.029
F 0.031 0.096 0.483 0.017 0.02 0.012 0.014 0.001 0.02 0.034 0.03 0.036*
G 0.023 0.014 0.012 0.01 0.007 0.014 0.014 0.014 0.016 0.01 0.027 0.011*
H 0.077 0.022 0.027 0.053 0.013 0.018 0.021 0.011 0.017 0.021 0.028 0.596*
Note: with table 2
11 fused cell screen of table selects (plate 10)
1 2 3 4 5 6 7 8 9 10 11 12
A 0.034 0.03 0.021 0.026 0.013 0.023 0.029 0.012 0.015 0.055 0.016 0.532
B 0.008 0.002 0.001 0 0 0.016 0.336 0.001 0.001 0.001 0.001 0.001
C 0.014 0.009 0.018 0.007 0.275 0.014 0.011 0.001 0.018 0.017 0.011 0.001
D 0.015 0.013 0.018 0.001 0.001 0.407 0.085 0.011 0.011 0.001 0.009 0.019
E 0.027 0.013 0.015 0.037 0.009 0.061 0.015 0.032 0.011 0.017 0.024 0.012
F 0.014 0.009 0.009 0.017 0.007 0.011 0.014 0.007 0.007 0.009 0.014 0.015*
G 0.023 0.013 0.011 0.011 0.008 0.014 0.014 0.015 0.015 0.052 0.47 0.012*
H 0.023 0.022 0.014 0.019 0.013 0.018 0.021 0.026 0.016 0.021 0.028 0.617*
Note: with table 2
Step 7) monoclonal cell two sieves
24 plants of positive cell strains that monoclonal primary dcreening operation is obtained use AGR2 albumen and label protein wrapper sheet, using the side ELISA respectively Method carries out programmed screening, further screens positive cell strain.
As a result as shown in table 12, AGR2 protein determination result is positive and label protein measurement result is that negative monoclonal is thin Born of the same parents' strain shares 5 plants, respectively 2B9,4F3,7G11,9F3 and 10G11.
Table 12 carries out ELISA screening with AGR2 albumen and label protein to 24 positive colonies
Number 1D6 1H2 2B9 4F3 5E12 6D1 6D6 6E1 6G1 6G2 6H2 7A3
AGR2 0.036 0.097 0.783* 0.653* 0.703 0.022 0.015 0.014 0.026 0.75 0.022 0.165
Label 0.072 0.129 0.039* 0.027* 0.843 0.041 0.039 0.032 0.039 0.481 0.051 0.15
Number 7C12 7G11 8D6 8E3 9A4 9A6 9F3 10A12 10B7 10C5 10D6 10G11
AGR2 0.033 0.225* 0.001 0.085 0.585 0.129 0.675* 0.001 0.684 0.091 0.001 0.46*
Label 0.068 0.039* 0.02 0.024 0.639 0.031 0.092* 0.031 0.9 0.029 0.038 0.077*
Control It is negative Blank It is positive
AGR2 0.043 0.018 0.453
Label 0.055 0.028 0.207
Note: label refers to label protein His；It is positive colony with *.
Step 8) monoclonal cell subgroup identification
Subgroup identification is carried out to 5 plants of positive cell strains that above-mentioned screening obtains.
(1) 100mM PBS(pH7.4 is used) dilution coated antibody is to 0.5 ug/ml, and every hole adds 0.1ml, and 4 DEG C are overnight；
(2) PBST is washed 2 times；200ul confining liquid is added in every hole, and 370C is incubated for 2h；
(3) PBST is washed 3 times；100ul hybridoma supematant is added in every hole, and 370C is incubated for 1h；
(4) PBST is washed 3 times；With the antibody 0.1ml of the diluted HRP label of confining liquid 1:1000 (κ, λ) or 1:2000 (others) Every hole is separately added into hole appropriate, 37 DEG C of incubation 1h；
(5) PBST is washed 3 times；Every hole adds 50 ul substrate solutions, and 10-20min is interior to survey light absorption value, record in dual wavelength (450,630) Save data.
Subgroup identification result is as shown in table 13, obtains 4 plants of IgG Type Positive hybridoma cell strains, be 2B9,4F3,9F3 and 10G11。
Table 13 carries out subgroup identification to 5 plants of positive cell strains
IgM IgG1 IgG2a IgG2b IgG3 IgGA κ λ
2B9 0.113 0.026 0.556 0.079 0.06 0.141 0.289 0.105
4F3 0.083 0.034 0.529 0.036 0.046 0.048 0.252 0.134
7G11 0.676 0.02 0.038 0.023 0.026 0.035 0.303 0.096
9F3 0.046 0.01 0.261 0.02 0.028 0.028 0.212 0.072
10G11 0.06 0.905 0.026 0.018 0.035 0.029 0.182 0.073
Negative control 0.034 0.001 0.035 0.02 0.024 0.027 0.03 0.039
Step 9) Western Blot and Transwell screen monoclonal cell strain
Experimental method is similar to previous experiments method in this example in this step, the difference is that being single used in this step Clone cell culture supernatant.Fig. 6 shows Western Blot testing result, and 2B9,4F3 and 10G11 are capable of detecting when HT29 cell In endogenic AGR2 albumen；Further, using these three monoclonal cell strains of Transwell experiment screening, Fig. 7 result Display 10G11 can obviously block exogenous AGR2 to the rush migration of SW48 cell.
The purifying of step 10) monoclonal antibody
(1) we select 2B9,10G11 hybridoma cell strain and are seeded in SPF grades of mouse peritoneals, extract mouse ascites after a week, Purified with Protein A/G；
(2) SDS-PAGE detects purified antibodies purity；
(3) ELISA detects antibody affinity costant.
Fig. 8 shows SDS-PAGE detection antibody purity as a result, two kinds of antibody are in heavy chain region (55KDa) and light chain area (25KDa) has clear band, it can be seen that antibody purity is greater than 90%；Table 14 shows antibody affinity costant testing result, can be with The affinity costant that 2B9 is calculated is 3.95E+09, and the affinity costant of 10G11 is 5.19E+08.
The affinity costant of 14 ELISA of table detection antibody
200 400 800 1600 3200 6400 12800 25600 51200 102400 204800 Blank
2B9 1.436 1.432 1.353 1.261 1.19 1.045 0.912 0.737 0.562 0.406 0.282 0.035
10G11 1.048 1.028 0.878 0.752 0.66 0.586 0.475 0.364 0.239 0.125 0.094 0.024
Note: 150000 x A/ antibody concentration of affinity costant ≈；Extension rate corresponding to the OD value of A=1/2.
The barrier effect of 4 monoclonal antibody of embodiment
Using the barrier effect of Transwell method detection monoclonal antibody, walked in experimental method and embodiment 3 in the present embodiment It is rapid 4) similar, the difference is that in the present embodiment under SW48 cell room be added be monoclonal antibody 2B9 after purification or 10G11 ；Express and secrete AGR2 additionally, due to HT29 cell height, thus we in the Transwell experiment of HT29 cell on Monoclonal antibody is added in room, and HT29 upper chamber cell number is 100,000, and aperture, fixed, dye are taken out after cultivating 72 hours in the incubator Color is simultaneously taken pictures.
As a result as shown in figure 9, two kinds of monoclonal antibodies 2B9 and 10G11 of purifying can block additional AGR2 albumen to draw The SW48 cell migration risen, but the barrier effect of 10G11 becomes apparent from, therefore that our subsequent experimental selections is 10G11；From Figure 10 further finds out that monoclonal antibody 10G11 can obviously block the rush migration of secreting type AGR2 in HT29 cell.
The specificity of 5 AGR2 monoclonal antibody of embodiment
1) Western Blot detects the specificity of monoclonal antibody
Experimental method is similar to step 4) in example 3 in this example, the difference is that primary antibody used in this example is purifying Antibody afterwards, and antibody dilution ratio is 1:1000.
As a result as shown in figure 11, monoclonal antibody 10G11 can detecte highly expressed AGR2 albumen in HT29 cell, and And degrees of specificity is higher, this is consistent with the testing result of mice serum in embodiment 3 and result reported in the literature.
) Immunofluorescence test monoclonal antibody specificity
(1) pancreatin digests colon cancer cell HT29；
(2) round coverslip is put into 24 orifice plates, PBS rinse is primary；
(3) postdigestive HT29 cell is transferred to 24 orifice plates；
(4) it after cell is adherent, inhales and abandons culture medium, PBS embathes twice；
(5) 4% paraformaldehyde room temperatures fix 15min, and PBS embathes three times；
(6) 0.2%TritonX-100 Cell-transmission model 15min, PBS embathe three times；
(7) the PBS room temperature containing 10%FBS closes 1h；
(8) control group adds IgG, and experimental group adds monoclonal antibody, 1:200 dilution, 4 DEG C of overnight incubations；
(9) PBS embathes 3 times, adds fluorescence secondary antibody, is incubated at room temperature 1h；
(10) PBS embathes 3 times, and DAPI dyes 20min, and PBS is observed and taken pictures under laser confocal microscope after embathing 2 times Record.
As a result as shown in figure 12, monoclonal antibody 10G11 can detecte the intracellular AGR2 of HT29, and control antibodies are examined It is negative to survey result, this has further demonstrated that the specificity of monoclonal antibody 10G11 in the present invention.
Sequence table
<110>Wuhan Union Hospital
<120>a kind of monoclonal antibody hybridoma cell strain and its application for AGR2
<160>2
<210>1
<211>528
<212>DNA
<213>people (Homo sapiens)
<221>CDS
<400>1
atggagaaaa ttccagtgtc agcattcttg ctccttgtgg ccctctccta cactctggcc 60
agagatacca cagtcaaacc tggagccaaa aaggacacaa aggactctcg acccaaactg 120
ccccagaccc tctccagagg ttggggtgac caactcatct ggactcagac atatgaagaa 180
gctctatata aatccaagac aagcaacaaa cccttgatga ttattcatca cttggatgag 240
tgcccacaca gtcaagcttt aaagaaagtg tttgctgaaa ataaagaaat ccagaaattg 300
gcagagcagt ttgtcctcct caatctggtt tatgaaacaa ctgacaaaca cctttctcct 360
gatggccagt atgtccccag gattatgttt gttgacccat ctctgacagt tagagccgat 420
atcactggaa gatattcaaa tcgtctctat gcttacgaac ctgcagatac agctctgttg 480
cttgacaaca tgaagaaagc tctcaagttg ctgaagactg aattgtaa 528
<210>2
<211>175
<212>PRT
<213>people (Homo sapiens)
<400>2
Met Glu Lys Ile Pro Val Ser Ala Phe Leu Leu Leu Val Ala Leu Ser
1 5 10 15
Tyr Thr Leu Ala Arg Asp Thr Thr Val Lys Pro Gly Ala Lys Lys Asp
20 25 30
Thr Lys Asp Ser Arg Pro Lys Leu Pro Gln Thr Leu Ser Arg Gly Trp
35 40 45
Gly Asp Gln Leu Ile Trp Thr Gln Thr Tyr Glu Glu Ala Leu Tyr Lys
50 55 60
Ser Lys Thr Ser Asn Lys Pro Leu Met Ile Ile His His Leu Asp Glu
65 70 75 80
Cys Pro His Ser Gln Ala Leu Lys Lys Val Phe Ala Glu Asn Lys Glu
85 90 95
Ile Gln Lys Leu Ala Glu Gln Phe Val Leu Leu Asn Leu Val Tyr Glu
100 105 110
Thr Thr Asp Lys His Leu Ser Pro Asp Gly Gln Tyr Val Pro Arg Ile
115 120 125
Met Phe Val Asp Pro Ser Leu Thr Val Arg Ala Asp Ile Thr Gly Arg
130 135 140
Tyr Ser Asn Arg Leu Tyr Ala Tyr Glu Pro Ala Asp Thr Ala Leu Leu
145 150 155 160
Leu Asp Asn Met Lys Lys Ala Leu Lys Leu Leu Lys Thr Glu Leu
165 170 175
Sequence table
<110>Wuhan Union Hospital
<120>a kind of monoclonal antibody hybridoma cell strain and its application for secreting anti-AGR2
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 528
<212> DNA
<213> Homo sapiens
<400> 1
atggagaaaa ttccagtgtc agcattcttg ctccttgtgg ccctctccta cactctggcc 60
agagatacca cagtcaaacc tggagccaaa aaggacacaa aggactctcg acccaaactg 120
ccccagaccc tctccagagg ttggggtgac caactcatct ggactcagac atatgaagaa 180
gctctatata aatccaagac aagcaacaaa cccttgatga ttattcatca cttggatgag 240
tgcccacaca gtcaagcttt aaagaaagtg tttgctgaaa ataaagaaat ccagaaattg 300
gcagagcagt ttgtcctcct caatctggtt tatgaaacaa ctgacaaaca cctttctcct 360
gatggccagt atgtccccag gattatgttt gttgacccat ctctgacagt tagagccgat 420
atcactggaa gatattcaaa tcgtctctat gcttacgaac ctgcagatac agctctgttg 480
cttgacaaca tgaagaaagc tctcaagttg ctgaagactg aattgtaa 528
<210> 2
<211> 175
<212> PRT
<213> Homo sapiens
<400> 2
Met Gly Leu Ile Pro Val Ser Ala Pro Leu Leu Leu Val Ala Leu Ser
1 5 10 15
Thr Thr Leu Ala Ala Ala Thr Thr Val Leu Pro Gly Ala Leu Leu Ala
20 25 30
Thr Leu Ala Ser Ala Pro Leu Leu Pro Gly Thr Leu Ser Ala Gly Thr
35 40 45
Gly Ala Gly Leu Ile Thr Thr Gly Thr Thr Gly Gly Ala Leu Thr Leu
50 55 60
Ser Leu Thr Ser Ala Leu Pro Leu Met Ile Ile His His Leu Ala Gly
65 70 75 80
Cys Pro His Ser Gly Ala Leu Leu Leu Val Pro Ala Gly Ala Leu Gly
85 90 95
Ile Gly Leu Leu Ala Gly Gly Pro Val Leu Leu Ala Leu Val Thr Gly
100 105 110
Thr Thr Ala Leu His Leu Ser Pro Ala Gly Gly Thr Val Pro Ala Ile
115 120 125
Met Pro Val Ala Pro Ser Leu Thr Val Ala Ala Ala Ile Thr Gly Ala
130 135 140
Thr Ser Ala Ala Leu Thr Ala Thr Gly Pro Ala Ala Thr Ala Leu Leu
145 150 155 160
Leu Ala Ala Met Leu Leu Ala Leu Leu Leu Leu Leu Thr Gly Leu
165 170 175

Claims (7)

1.一种分泌抗AGR2蛋白的单克隆抗体的杂交瘤细胞株，所述杂交瘤细胞株的分类命名为：小鼠杂交瘤细胞株，该细胞株于2018年08月28日保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏中心登记入册保藏编号为CGMCC No.16288。1. A hybridoma cell line that secretes a monoclonal antibody against AGR2 protein, the classification of the hybridoma cell line is named: mouse hybridoma cell line, and the cell line was preserved in China Microorganisms on August 28, 2018 The Ordinary Microbiology Center of the Culture Collection Management Committee, and the deposit number of the collection center is CGMCC No.16288. 2.一种能特异性地识别、靶向AGR2蛋白的单克隆抗体，其特征在于：命名为单克隆抗体10G11，由权利要求1所述杂交瘤细胞株分泌得到。2 . A monoclonal antibody capable of specifically recognizing and targeting AGR2 protein, characterized in that: it is named as monoclonal antibody 10G11, which is secreted from the hybridoma cell line of claim 1 . 3.权利要求1所述杂交瘤细胞株在制备AGR2蛋白检测或诊断的药物或试剂中的应用。3. The application of the hybridoma cell line of claim 1 in the preparation of drugs or reagents for AGR2 protein detection or diagnosis. 4.权利要求1所述杂交瘤细胞株在制备治疗癌症的药物或试剂中的应用。4. The application of the hybridoma cell line of claim 1 in the preparation of a drug or reagent for treating cancer. 5.权利要求2所述单克隆抗体在制备AGR2蛋白检测或诊断的药物或试剂中的应用。5. The application of the monoclonal antibody of claim 2 in the preparation of drugs or reagents for AGR2 protein detection or diagnosis. 6.权利要求2所述单克隆抗体在制备治疗癌症的药物或试剂中的应用。6. The application of the monoclonal antibody of claim 2 in the preparation of a drug or reagent for treating cancer. 7.权利要求2所述单克隆抗体的制备方法，包括以下步骤：7. the preparation method of the described monoclonal antibody of claim 2, comprises the following steps: 1）重组表达载体的构建1) Construction of recombinant expression vector 根据AGR2开放阅读框序列，设计引物扩增AGR2编码区，通过EcoRI和XhoI酶切位点插入到pET28a载体中，构建pET28a-AGR2-His重组表达载体；According to the AGR2 open reading frame sequence, primers were designed to amplify the AGR2 coding region, and inserted into the pET28a vector through the EcoRI and XhoI restriction sites to construct the pET28a-AGR2-His recombinant expression vector; 2）AGR2重组蛋白的表达和纯化2) Expression and purification of AGR2 recombinant protein 将步骤1）中构建好的重组表达载体转化至BL21感受态大肠杆菌中，IPTG诱导蛋白表达，超声裂解细菌，离心获得蛋白上清；Transform the recombinant expression vector constructed in step 1) into BL21 competent E. coli, induce protein expression with IPTG, lyse the bacteria by ultrasonic, and obtain the protein supernatant by centrifugation; 通过镍柱亲和层析柱纯化，获得纯化的AGR2蛋白；Purified by nickel column affinity chromatography to obtain purified AGR2 protein; 3）单克隆抗体的筛选与制备3) Screening and preparation of monoclonal antibodies 将步骤2)中纯化的AGR2蛋白免疫ICR雌鼠，经过初次免疫和加强免疫后，取小鼠的脾细胞和SP2/0骨髓瘤细胞融合，有限稀释法获得单克隆；通过ELISA方法筛选出阳性杂交瘤细胞株，得到能够分泌针对AGR2蛋白的单克隆抗体的杂交瘤细胞株，命名为10G11，亚型鉴定为IgG1型；将杂交瘤细胞株接种至小鼠腹腔，收集小鼠腹水，通过Protein A/G纯化抗体。The AGR2 protein purified in step 2) was immunized with ICR female mice, and after the primary immunization and booster immunization, the spleen cells of the mice were fused with SP2/0 myeloma cells, and the monoclonal was obtained by the limiting dilution method; the positive cells were screened by ELISA method Hybridoma cell line was obtained to obtain a hybridoma cell line capable of secreting monoclonal antibodies against AGR2 protein, named 10G11, and the subtype was identified as IgG1 type; the hybridoma cell line was inoculated into the abdominal cavity of mice, and the ascites fluid of the mice was collected. A/G purified antibody.
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