CN110438088A - The preparation of 1 monoclonal antibody of lung cancer associated proteins and purification process - Google Patents
The preparation of 1 monoclonal antibody of lung cancer associated proteins and purification process Download PDFInfo
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Abstract
The present invention relates to protein engineering fields, and in particular to a kind of 1 monoclonal antibody of lung cancer associated proteins, preparation method and purification process, the preparation and purification method include: that immune mouse prepares 1 monoclonal antibody mouse ascites of lung cancer associated proteins；It is saltoutd using saturated ammonium sulfate, affinity chromatography two-step method antibody purification；Antibody saves.The 1 monoclonal antibody purity of lung cancer associated proteins obtained using preparation and purification method of the present invention reaches 99% through SDS-PAGE identification.The present invention provides preparation and the purifying process of a kind of 1 monoclonal antibody of lung cancer associated proteins.1 monoclonal antibody of lung cancer associated proteins produced by the present invention be liquid condition, 2-8 DEG C can long-term preservation, at room temperature can be reserved for 2 years.
Description
Technical field
The present invention relates to protein engineering fields, and in particular to the preparation method of 1 monoclonal antibody of lung cancer associated proteins with
And purifying process.
Background technique
Lung cancer associated proteins 1 (Overexpressed In Lung Cancer, OLC1) are a kind of new tumor-related
Because of the albumen of coding, 334 amino acid of overall length, Genbank ID:9798.Bioinformatic analysis discovery, lung cancer correlation egg
White OLC1 can activate NF-kB access in reporter gene level.In early stage (atypical hyperplasia and the original position that lung squamous cancer occurs
Cancer), the expression quantity of OLC1 increases, and prompts it in close relations with the early carcinomatous change of lung cancer.Some researches show that OLC1 exists recently
A variety of human malignancies, such as expression high in nasopharyngeal carcinoma, lung cancer, breast cancer, oophoroma, colorectal cancer and the cancer of the esophagus.
The common method for producing a large amount of monoclonal antibodies at present has mouse ascites preparation, big bottle culture and doughnut reaction
Three kinds of device, the former is chiefly used in laboratory preparation, both rear to be suitable for the factorial production.Ascites preparation method is at low cost, easy purification,
Purified antibodies concentration is high.The supernatant volume that big bottle culture obtains is big, but antibody concentration is low, toxigenic capacity and antibody purification cost
It is higher.Relative to big bottle culture, carrying out monoclonal antibody production using hollow fiber reactor is both economical method, but
The expense investment of apparatus is larger.
At present on the market without the related in-vitro diagnosis detection kit of lung cancer associated proteins 1.For kit,
High-purity, high-titer antibody preparation and purifying be that kit researches and develops successful key element, the Dan Ke which obtains
Grand antibody is that the research and development of later 1 detection kit of lung cancer associated proteins provide the foundation.
Summary of the invention
The present invention provides a kind of anti-lung cancer associated protein 1 monoclonal antibody, hybridoma cell strain and preparation method thereof, institutes
Antibody is stated with high degree of specificity, is used for specific recognition lung cancer associated proteins 1.
A kind of hybridoma cell strain for secreting anti-lung cancer associated protein 1 monoclonal antibody, number is OLC1-38 and OLC1-
15。
The present invention provides a kind of preparation method of hybridoma cell strain, step includes:
(1) the Balb/c mouse of 5~6 week old is immunized with 1 overall length antigen of lung cancer associated proteins；
(2) splenocyte of immune mouse is collected, and is merged with Sp2/0 myeloma cell, the hybridoma of fusion
Selective training is carried out with the methylcellulose semisolid selective medium containing hypoxanthine, methopterin and thymidine
It supports；
(3) Western blot (Western Blot) evaluation and screening positive hybridoma cell strain is used.
As a further preference, step (1) it is described it is immune include: by 1 overall length antigen of lung cancer associated proteins and isometric
Freund's complete adjuvant be sufficiently mixed emulsification, later using incomplete Freund's adjuvant emulsify antigen, be fully mixed to Water-In-Oil shape
It carries out subcutaneous multiple spot to 5~6 week old female Balb/c mouse after state to be immunized, 2~3 booster immunizations.
As a further preference, the fusion includes: to take mouse spleen that cell suspension is made under aseptic condition, in poly- second
Glycol mediates lower fusion mouse boosting cell and Sp2/0 myeloma cell, and after fusion plus IMDM cell culture medium terminates.
As a further preference, the mouse boosting cell and the number ratio of Sp2/0 myeloma cell are 5:1~10:1.
As a further preference, the methylcellulose containing hypoxanthine, methopterin and thymidine half
Include: in solid selective medium containing mass concentration be 1.25% methylcellulose, volumetric concentration be 25% fetal calf serum
With the IMDM culture medium of 2%HAT.
As a further preference, the screening includes: with Western blot (Western Blot), with transfection
The H1299 cell protein of 1 gene of lung cancer associated proteins is antigen, to transfect the H1299 cell protein of empty carrier for control, with anti-
1 monoclonal antibody of lung cancer associated proteins is measuring samples, screens positive hybridoma cell strain.
The present invention also protects the hybridoma cell strain prepared by above-mentioned preparation method.
The present invention also provides a kind of monoclonal antibodies, are produced by the hybridoma cell strain or the secretion of its passage cell strain
It gives birth to or is obtained through in vitro culture mode.
As a further preference, the hypotype of the monoclonal antibody is respectively IgG2a and IgG1 type.
The present invention provides the foundation of the purifying process of 1 monoclonal antibody of lung cancer associated proteins.
It is 99% that the 1 monoclonal antibody SDS-PAGE of lung cancer associated proteins prepared in this way, which detects its purity,；Relatively
Molecular weight is that heavy chain 55kD adds light chain 25kD.
Further, the technical scheme comprises the following steps for purifying:
(1) the 1 monoclonal antibody mouse ascites of lung cancer associated proteins of immune mouse preparation are taken；
(2) it successively saltouts through supersaturated ammonium sulfate, the purifying of affinity chromatography two-step method obtains antibody；
(3) by antibody filtration sterilization, it is stored in 2-8 DEG C of refrigerator.
Preferably, step (2) specifically includes the following steps:
(2-1) dilution: the mouse ascites that step (1) is obtained dilute 1 times with phosphate buffer；
(2-2) saltouts: by after dilution sample and saturated ammonium sulfate volume ratio 1:1 mix, 2-8 DEG C stands overnight, then passes through
2000~6000rpm is centrifuged 30min, discards supernatant, and dialysis is in 0.1M phosphate after precipitating 0.1M phosphate buffer dissolution
10~14h in buffer；
(2-3) affinity chromatography: after saltouing sample using affinitive layer purification chromatographic column successively through balance, loading, again put down
Antibody purification sample is obtained after weighing apparatus, elution；
(2-4) dialysis: by the dialysis of purified antibodies sample in dialyzate, dialyzate used is pH7.2~7.4, concentration
The phosphate buffer of 0.1M.
Further, step (2-3) balance and the flow velocity of rebalancing setting are 0.5~1mL/min, use 3-5
The equilibrium liquid of a column volume, equilibrium liquid used be pH be 7.2~7.4, the phosphate buffer of 0.1M, buffer by sodium chloride,
Disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride composition.
Further, step (2-3) the affinitive layer purification chromatographic column is antibody affinity chromatography gel column.
Further, step (2-3) loading be the mouse ascites that will dilute after 0.22 μm of membrane filtration with
The speed loading of 0.2~1ml/min.
Further, flow velocity is set as 0.5~1mL/min when step (2-3) described elution, washes again after collecting eluting peak
3~5 column volumes are taken off, eluent pH used is 2.5~3.0, is made of glycine and hydrochloric acid, wherein the concentration of glycine is
0.1M。
Preferably, step (3) specifically includes the following steps:
Albumen sample of the dialysis in dialyzate is taken out, deposits in suitable container, is removed with 0.22 μm of membrane filtration
Bacterium is saved and 2-8 DEG C with sterile EP pipe packing.
Another object of the present invention is to provide prepare by the purification process of above-mentioned 1 monoclonal antibody of lung cancer associated proteins
1 monoclonal antibody of lung cancer associated proteins.
The beneficial effects of the present invention are:
Methylcellulose semisolid culturemedium colonized culture hybridoma is easier than limiting dilution assay many, first
It is that fused cell is separated from each other, is in colony growth, does not generate interference mutually, do not mix.The separation of monoclonal can be completed in one step
Work, greatly reduces workload, significant to shorten experimental period.
The 1 monoclonal antibody purity of lung cancer associated proteins obtained using preparation and purification method of the present invention is reflected through SDS-PAGE
Surely reach 99%.
1 monoclonal antibody of lung cancer associated proteins produced by the present invention be liquid condition, 2-8 DEG C can long-term preservation, in room temperature
It can be reserved for 2 years down.
Detailed description of the invention
Fig. 1 is 1 monoclonal antibody SDS-PAGE testing result figure of lung cancer associated proteins: where swimming lane M: albumen Marker；
Swimming lane 1: 1 monoclonal antibody of lung cancer associated proteins after purification；A is heavy chain 55kD, and B is light chain 25kD.
Fig. 2 is 1 monoclonal antibody affinity chromatography purification result figure of lung cancer associated proteins, and wherein C is eluting peak.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified certainly
What routine biochemistry reagent shop was commercially available.
Embodiment one secretes the preparation of the hybridoma cell strain of anti-lung cancer associated protein 1 monoclonal antibody
Preparation method includes the following steps:
1. animal immune
First immunisation, 1 antigen of lung cancer associated proteins is mixed and is emulsified with isometric Freund's complete adjuvant, fully emulsified
It is carried out subcutaneously by the antigenic content of every 15~30 μ g of mouse to 5~6 week old female Balb/c mouse after to the state of Water-In-Oil
Multiple spot is immune；Booster immunization is carried out after 2 weeks, antigen and isometric incomplete Freund's adjuvant emulsify, and carry out 2~3 reinforcements altogether and exempt from
Epidemic disease, 2 weeks each immunization interval periods complete 3 times after being immunized, and the 7th day eye socket blood sampling detection serum antibody titer takes mice serum
Potency is detected with indirect elisa method.The antigen of immunizing dose is directly dissolved in PBS solution after 1 week after potency is higher than 1:10000
In, abdominal cavity impact is carried out to mouse and is immunized, can be merged after 3 days.
2. cell fusion
After mouse peritoneal impact is 3 days immune, extracts eyeball dislocation of cervical vertebra and put to death, and collect positive control serum, take out spleen
It is dirty, it is prepared into single cell suspension, the Sp2/0 myeloma cell in logarithmic growth phase is then taken, is mixed in a certain ratio (5:1
~10:1), it after the polyethylene glycol of 50% volume acts on 1min, is terminated with IMDM cell culture medium, after low-speed centrifugal, is used
Methylcellulose semisolid selective medium containing HAT is resuspended, and after mixing well, spreads ware, is placed in 5%CO2, in 37 DEG C of incubators
Culture.Specific step is as follows:
1) splenocyte suspension: after the Balb/c mouse dislocation of cervical vertebra execution for reaching potency after being immunized, aseptic condition is lower to be beaten
It opens abdominal cavity and takes out spleen, prepare single splenocyte suspension using dismembyator and sieve, strict guarantee is wanted in whole preparation process
It is sterile.Then splenocyte suspension is counted；
2) Sp2/0 myeloma cell: Sp2/0 myeloma cell recovers for 2 weeks before fusion, uses 8-anaguanine culture medium
It selects culture to change into after a week to continue to cultivate for 10% IMDM culture medium containing mass concentration, fusion the previous day passes on it training
It supports, is at logarithmic growth phase；
3) fused cell: splenocyte and Sp2/0 myeloma cell contain 50% volume in 0.7mL with the ratio of 5:1~10:1
Polyethylene glycol mediate lower fusion, after 37 DEG C of culture 1min, 10mL IMDM cell culture medium, which terminates, to be merged, low-speed centrifugal
Remove polyethylene glycol；
4) selectivity culture: the methylcellulose half with 40mL containing hypoxanthine, methopterin and thymidine is solid
Cell is resuspended in body selective medium, after mixing of turning upside down, is transferred in 35mL plate, every plate about 2mL, in 5%CO2,37
It is cultivated in DEG C incubator.It cultivates 7 days rear clones and is transferred to 96 orifice plates, be added and contain the fetal calf serum of 15% volume and containing for 2% volume
Continue to cultivate in the IMDM cell culture medium of HT (hypoxanthine and thymidine).When cell fusion degree reaches 50~
70%, collect culture medium supernatant.
3. positive hole screening
Western blot (Western Blot) screening antibodies: respectively will transfection 1 gene of lung cancer associated proteins and sky
The H1299 cell of carrier extracts total protein, every 40 μ g loading of hole, through sodium dodecyl sulfate-polypropylene acrylamide gel electricity respectively
Swimming is separated.Albumen is gone into PVDF membrane after electrophoresis, skim milk closes 1h, and (anti-lung cancer is related for primary antibody
1 hybridoma culture supernatant 1:5 of albumen dilution) 4 DEG C be incubated overnight.After washing buffer buffer (PBST) washes film 3 times, secondary antibody room
Temperature is incubated for 1h, washes exposure after film, analyzes result.
4. the foundation of monoclonal cell strain
2 subclones are carried out to the cell strain of detection antibody positive, the stably excreting that the subclone stage is filtered out is positive
The monoclonal cell strain of antibody expands culture, and freezes conservation.Must recover same batch after cell strain freezes
In one identified, standard are as follows: Xi Bao Shuo≤1,000,000 cell living of 1. recovering/；2. vibrant cell in living cells
≤ 50 ten thousand/strains；3. cannot have other microorganisms in addition to cell strain cell in recovery cell (such as: bacterium, fungi, mycoplasma
Deng) occur；4. recovery cell, which grows into after certain amount to select the cell grown and make monoclonal, counts bed board, and detects Dan Ke
The whether full sun of grand secretory antibody ability has antibody-secreting.It chooses this plant of cell and carries out subsequent experimental.And it is this plant of hybridoma is thin
Born of the same parents send to China Committee for Culture Collection of Microorganisms's common micro-organisms center's preservation, deposit number are as follows: 15587 (OLC1-15),
15588(OLC1-38)。
Embodiment two prepares anti-lung cancer associated protein 1 monoclonal antibody
(1) in vitro culture of anti-lung cancer associated protein 1 monoclonal antibody
The hybridoma that embodiment 1 is obtained is with IMDM cell culture medium (containing 10% fetal calf serum) in 5%CO2、37
It is cultivated in DEG C incubator, after cell enters logarithmic growth phase, collects cell conditioned medium, be used for simple preliminary identification.
(2) antibody subtype is identified
It is detected by IsoStrip Mouse Monoclonal Antibody Isotyping Kit (Roche), this hair
Two plants of bright produced antibody subtypes of cell are respectively IgG2a (OLC1-38) and IgG1 (OLC1-15) type.
The foundation of the monoclonal antibody-purified technique of three lung cancer associated proteins of embodiment 1
1. dilution: the mouse ascites of acquisition are diluted with 0.2M phosphate buffer by 1:1；
2. saltouing: by after dilution sample and saturated ammonium sulfate volume ratio 1:1 mix, 2-8 DEG C stands overnight, then through 2000
~6000rpm is centrifuged 30min, discards supernatant, and dialysis is in 0.1M phosphate is slow after precipitating 0.1M phosphate buffer dissolution
10~14h；
3. affinitive layer purification prepares: affinitive layer purification instrument is full-automatic purifying instrument, affinitive layer purification institute
It is antibody affinity chromatography gel column with material；
4. affinitive layer purification chromatographs column equilibration: flow velocity is set as 0.5~1ml/min, and balancing equilibrium liquid used is 3~5
A column volume, equilibrium liquid used be pH be 7.2~7.4, the phosphate buffer of 0.1M, buffer is by sodium chloride, phosphoric acid hydrogen two
Sodium, potassium dihydrogen phosphate, potassium chloride composition；
5. affinitive layer purification loading: by the mouse ascites diluted with 0.2~1ml/min after 0.22 μm of membrane filtration
Speed loading；
6. affinitive layer purification chromatographic column rebalancing: flow velocity is set as 0.5~1ml/min, balance equilibrium liquid used be 3~
5 column volumes, equilibrium liquid used be pH be 7.2~7.4, the phosphate buffer of 0.1M, buffer is by sodium chloride, phosphoric acid hydrogen two
Sodium, potassium dihydrogen phosphate, potassium chloride composition；
7. affinitive layer purification chromatographic column elutes: flow velocity is set as 0.5~1ml/min, collects eluting peak (such as Fig. 2), receives
3~5 column volumes are eluted again after collection eluting peak, purifying eluent pH used is 2.5~3.0, it is made of glycine and hydrochloric acid,
The concentration of middle glycine is 0.1M；
8. affinitive layer purification chromatographic column saves: being full of instrument pipeline with 20% ethyl alcohol；
9. purifying gained antibody sample dialysis: by the dialysis of purified antibodies sample in dialyzate, dialyzate used is
PH7.2~7.4, concentration 0.1M phosphate buffer；
10. albumen sample of the dialysis in dialyzate is taken out, deposit in suitable container, with 0.22 μm of membrane filtration
Degerming is stored in 2-8 DEG C with sterile EP pipe packing.
The SDS-PAGE of lung cancer associated proteins 1 is detected:
(a) SDS-PAGE electrophoresis: 1 sample SDS-PAGE electrophoresis of lung cancer associated proteins will be purified, will first be configured before loading
Polyacrylamide gel be attached on vertical electrophoresis apparatus, with 50V constant pressure sky run 30min；
(b) coomassie brilliant blue staining: gel is placed in Coomassie brilliant blue dye liquor and dyes 30min, is washed away with destainer
Background；
(c) result: identify that purity is 99%, wherein heavy chain 55kD, light chain 25kD (such as Fig. 1).
1 protein content determination of lung cancer associated proteins: albumen is detected to above-mentioned monoclonal antibody after purification Lowry method
Concentration, protein content 5.40mg/mL.
Embodiment described above is only that preferred embodiments of the present invention will be described, not to model of the invention
It encloses and is defined, without departing from the spirit of the design of the present invention, those of ordinary skill in the art are to technical side of the invention
The various changes and improvements that case is made, should fall within the scope of protection determined by the claims of the present invention.
Claims (9)
1. secreting the hybridoma cell strain of anti-lung cancer associated protein 1 monoclonal antibody, deposit number is respectively 15587 Hes
15588。
2. the anti-lung cancer associated protein 1 monoclonal antibody prepared by hybridoma cell strain described in claim 1.
3. the purification process of anti-lung cancer associated protein 1 monoclonal antibody as claimed in claim 2, which is characterized in that including following
Step:
(1) the 1 monoclonal antibody mouse ascites of lung cancer associated proteins of immune mouse preparation are taken；
(2) it successively saltouts through supersaturated ammonium sulfate, the purifying of affinity chromatography two-step method obtains antibody；
(3) by antibody filtration sterilization, it is stored in 2-8 DEG C of refrigerator.
4. purification process as claimed in claim 3, which is characterized in that step (2) specifically includes the following steps:
(2-1) dilution: the mouse ascites that step (1) is obtained dilute 1 times with phosphate buffer；
(2-2) saltouts: by after dilution sample and saturated ammonium sulfate volume ratio 1:1 mix, 2-8 DEG C stands overnight, then through 2000
~6000rpm is centrifuged 30min, discards supernatant, and dialysis is in 0.1M phosphate-buffered after precipitating 0.1M phosphate buffer dissolution
10~14h in liquid；
(2-3) affinity chromatography: after saltouing sample using affinitive layer purification chromatographic column successively through balance, loading, rebalancing, wash
Antibody purification sample is obtained after de-；
(2-4) dialysis: by the dialysis of purified antibodies sample in dialyzate, dialyzate used is pH7.2~7.4, concentration 0.1M
Phosphate buffer.
5. purification process as claimed in claim 4, it is characterised in that: the flow velocity of step (2-3) balance and rebalancing setting
For 0.5~1mL/min, using the equilibrium liquid of 3~5 column volumes, equilibrium liquid used be pH be 7.2~7.4, the phosphate of 0.1M
Buffer, buffer are made of sodium chloride, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium chloride.
6. purification process as claimed in claim 4, it is characterised in that: step (2-3) the affinitive layer purification chromatographic column is anti-
Body affinity chromatography gel column.
7. purification process as claimed in claim 4, it is characterised in that: step (2-3) loading is the mouse abdomen that will be diluted
Water is after 0.22 μm of membrane filtration with the speed loading of 0.2~1ml/min.
8. purification process according to claim 4, it is characterised in that: flow velocity is set as 0.5 when step (2-3) described elution
~1mL/min elutes 3~5 column volumes after collecting eluting peak again, and eluent pH used is 2.5~3.0, by glycine and salt
Acid composition, wherein the concentration of glycine is 0.1M.
9. purification process as claimed in claim 4, which is characterized in that step (3) will be specifically includes the following steps: will dialyse in dialysis
Albumen sample in liquid takes out, and deposits in suitable container, with 0.22 μm of membrane filtration degerming, is dispensed and is protected with sterile EP pipe
It is stored in 2-8 DEG C.
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Citations (3)
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|---|---|---|---|---|
| WO1993006858A1 (en) * | 1991-10-09 | 1993-04-15 | Dana Farber Cancer Institute, Inc. | Lung cancer-associated protein |
| CN1556210A (en) * | 2004-01-01 | 2004-12-22 | 昆明医学院第一附属医院 | Human protein phosphokinase 2A point mutanted lung cancer related antigen gene |
| CN101885771A (en) * | 2009-05-14 | 2010-11-17 | 蒋芳林 | Preparation and applications of monoclonal antibody and polyclonal antibody of human tissue factor pathway inhibitor-2 |
-
2019
- 2019-08-16 CN CN201910760076.7A patent/CN110438088A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1993006858A1 (en) * | 1991-10-09 | 1993-04-15 | Dana Farber Cancer Institute, Inc. | Lung cancer-associated protein |
| CN1556210A (en) * | 2004-01-01 | 2004-12-22 | 昆明医学院第一附属医院 | Human protein phosphokinase 2A point mutanted lung cancer related antigen gene |
| CN101885771A (en) * | 2009-05-14 | 2010-11-17 | 蒋芳林 | Preparation and applications of monoclonal antibody and polyclonal antibody of human tissue factor pathway inhibitor-2 |
Non-Patent Citations (3)
| Title |
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| 张坤鹏等: "OLC1在肺鳞癌组织中的过表达与患者的不良预后相关", 《中国肺癌杂志》 * |
| 杨龙海等: "OLC1蛋白在非小细胞肺癌患者外周血血浆中的水平及其临床意义", 《中华肿瘤杂志》 * |
| 王云双等: "《临床免疫学检验》", 31 March 2009, 北京：军事医学科学出版社 * |
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