CN108384747A - Express the Chinese hamster ovary celI serum free suspension cultural method of Rabies virus antibody - Google Patents
Express the Chinese hamster ovary celI serum free suspension cultural method of Rabies virus antibody Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
- C12N5/0682—Cells of the female genital tract, e.g. endometrium; Non-germinal cells from ovaries, e.g. ovarian follicle cells
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- C12N2500/00—Specific components of cell culture medium
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- C12N2500/24—Iron; Fe chelators; Transferrin
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/33—Insulin
Abstract
The invention discloses a kind of Chinese hamster ovary celI serum free suspension cultural methods of expression Rabies virus antibody.This method includes the culture of cell strain, the collection of cell and serum free medium preparation, the pretreatment of culture dish, cell culture and the observation of cell state totally 4 steps.The present invention selects common mammalian cell expression system Chinese hamster ovary celI to study, and the method by improving experiment determines the condition of serum free suspension culture；Using free serum culture cell, production cost can be greatly reduced, reduces the pollution of foreign protein；The expression quantity of antibody protein is improved, and is cultivated in serum-free and is easy purifying destination protein, improves production efficiency.
Description
Technical field
The present invention relates to a kind of Chinese hamster ovary celI serum free suspension cultural method of expression Rabies virus antibody, and the CHO of optimization are thin
Born of the same parents' serum free medium.
Background technology
Antibody engineering technology especially exists with the main force that the development of modern biotechnology has been biotechnology industry
Biotechnological pharmaceutics field occupies an important position.Antibody be biomedical sector purposes it is most commonly used have treatment tumour and
The protein product of major disease, and treatment of the antibody as drug for human diseases possesses very long history.Currently, lactation is dynamic
There is object cell extremely important application value, advantage to be that the weight with humanized can be expressed in the production of biological products
Group protein product (Rodrigues et al.2013) that is therapeutic and maintaining bioactivity.Wherein, generation the most typical
Table is to use Chinese hamster ovary cell expression antibody, and expressing cho cell system has the advantages that large-scale production, specific manifestation
For can high efficiency stable expression destination protein, while there is easy-operating characteristic.In order to obtain a large amount of antibody protein, Chinese storehouse
Mouse gonad cell (CHO) is used for expressing foreign protein by most researchers, due to lacking DHFR- genes, is easy screening purpose
Cell strain is a kind of ideal expression system (Gandor et al.1995；Jun et al.2005).
With the rise of biological products industry in recent years, Chinese hamster ovary celI has been widely used for expression antibody and other diseases
Malicious glycoprotein product.The product requirement of bioprotein class can in corresponding cell great expression, be easy purifying, it is at low cost and
Foreign protein and pollutant albumen that may be present will minimize standard.Traditional Chinese hamster ovary celI can only contain serum
Adherent growth in culture medium, the serious expression quantity for limiting albumen, because there are certain limitations for the cell of adherent growth, together
When can all introduce foreign protein using serum.The ingredient of serum is very abundant and disclosure satisfy that the growth of most cells needs
It asks, but after using serum, undoubtedly increases production cost and be easily introduced external source contaminating protein substance (Rodrigues et
Al.2013), exactly these disadvantages require the enterprise of production biological products and research institution urgently to find one kind and cell can be allowed to exist
It is grown in the state of serum-free and suspension, can thus reduce the pollution of foreign protein, reduce cost, so as to realize big rule
Mould produces (Chen et al.2000).
On the basis of this laboratory research, mammalian cell CHO is built to express the antibody protein of mad dog, and sieve
The cell strain of one plant of expression antibody protein is selected.In order to overcome the technical barrier of serum-free and the culture that suspends, experiment of the invention
Method can primarily determine Chinese hamster ovary celI in serum free medium can normal growth, while there is the ability of suspension growth.
Currently, the expression of antibody protein mostly uses mammalian cell expression system, but the growth of mammalian cell
It is more stringent, it is necessary to be grown in the culture medium containing serum, therefore increase the cost of production and the risk of pollution.Thus
The method of the present invention can overcome the above-mentioned problem that Chinese hamster ovary celI occurs when expressing antibody, be provided for the large-scale production of antibody
Ideal experiment basis.
Invention content
The technical problem to be solved in the present invention is to provide a kind of Chinese hamster ovary celI serum free suspension culture sides of expression Rabies virus antibody
Method.
The invention solves another technical problem be to provide a kind of Chinese hamster ovary celI serum free medium of optimization.
For Chinese hamster ovary celI serum free suspension cultural method, the technical solution adopted by the present invention is to express Rabies virus antibody
Chinese hamster ovary celI serum free suspension cultural method, includes the following steps：
(1) culture of cell strain：The cell strain is Chinese hamster ovary cell, and hereinafter referred to as cell；By cell
With 2 × 105~6 × 105The quantity of cells/ml is inoculated in blood serum medium, and condition of culture is 37 DEG C, 5.0% carbon dioxide
Incubator obtains cell A；It measures the growth curve of cell A and the growth song of cell A after micro somatomedin is added
Line；The somatomedin is growth accelerator G-CSF-RCHP, and is added to blood serum medium by 0.2~0.4 μ L/ml；
The blood serum medium is the IMDM liquid solutions containing 10% fetal calf serum；
(2) collection of cell：The cell A that will be grown in blood serum medium after handling 1~5min using pancreatin, is collected
For cell A in the centrifuge tube of 15ml, 1000rpm centrifuges 3min, sucks the 10ml free serum culture based sols washing of 37 DEG C of preheatings
Cell is primary, and final constant volume obtains cell B, and using Trypan Blue dyeing meters in the serum free medium of volume 5ml
Number cell B, measures the quantity of cell B；
The serum free medium is formulated by following components：
DMEM/F12 liquid solution 1ml, and filtration sterilization adjust pH to 7.0~7.2；
2~10ng/ml of Trace selenium；
Insulin 1-3 μ g/ml；
2~15 μ g/ml of transferrins；
Growth accelerator G-CSF-RCHP：0.2~0.4 μ l/ml；
Anti-cell nodule agent：0.1~0.5 μ l/ml；
And after the completion of the serum free medium is prepared, use preceding 37 DEG C of pre- stand-by heats；
(3) pretreatment of 6 orifice plates：It is 7.0~7.2 1 × BS solution to take pH value, and diluted concentration is the fibrous strands of 1mg/ml
Albumen is connect, final working concentration is obtained and is the mixed solution of 20~80 μ g/ml, then draws 1ml mixed solutions and 6 orifice plates, place is added
Manage the time be 15~60min to get to pretreatment 6 orifice plates；
(4) cell culture：Cell B is pressed 2 × 105~6 × 105In the quantity inoculation pretreatment 6 orifice plates of cells/ml, then
By cell B successively in the IMDM fluid nutrient mediums containing 5% fetal calf serum, the IMDM Liquid Cultures containing 2.5% fetal calf serum
By generation in base, the IMDM fluid nutrient mediums containing 1.25% fetal calf serum, the IMDM fluid nutrient mediums containing 0.1% fetal calf serum
Culture, when cell B is full of adherent growth or after culture overnight growth, you can replace with above-mentioned fetal calf serum ratio and gradually reduce
IMDM fluid nutrient mediums, until replace with serum free medium；Cell B by micro- sem observation Jing Guo culture overnight simultaneously
Form, and according to the state status of cell B growth, when cell B is gradually converted to suspension growth, and cell shape by adherent growth
State is converted to circle by arc or fusiformis, the not pretreated 6 orifice plates of cell B switchings of suspension is continued to cultivate, finally from life
Suspension cell is screened in long cell B, and the suspension cell antibody expression filtered out is detected.
Preferably, in step (1), cell is with 2 × 105The quantity of cells/ml is inoculated in blood serum medium.
Preferably, in step (4), cell B presses 2 × 105The quantity inoculation pretreatment 6 orifice plates of cells/ml.
Preferably, the incubation time of cell B is 3~5 days in step (4).
Preferably, in step (4), using Western-Blotting detection mode to cell B express antibody into
Row detection.
For Chinese hamster ovary celI serum free medium, the technical solution adopted by the present invention is, a kind of Chinese hamster ovary celI of optimization is without blood
Clear culture medium, it is composed of the following components：
DMEM/F12 liquid solutions：1ml, filtration sterilization, pH to 7.0~7.2；
Trace selenium：2~10ng/ml；
Insulin：1~3 μ g/ml；
Transferrins：2~15 μ g/ml；
Growth promotion dosage G-CSF-RCHP：0.2~0.4 μ l/ml；
Anti-cell nodule agent：0.1~0.5 μ l/ml.
The beneficial effects of the invention are as follows：
1, common mammalian cell expression system Chinese hamster ovary celI is selected to be studied, the method by improving experiment, really
Determine the condition of serum free suspension culture；
2, free serum culture cell can greatly reduce production cost, reduce the pollution of foreign protein；
3, the expression quantity of antibody protein is improved, and is cultivated in serum-free and is easy purifying destination protein, improves production efficiency.
Specific implementation mode
Embodiment 1：Serum free medium
Serum free medium uses DMEM/F12, does not contain fetal calf serum, and trace element and transferrins is added, and adds
Enter micro growth growth-promoting agent, free serum culture based component refers to table 1.
After the completion of serum free medium is prepared, preceding 37 DEG C of pre- stand-by heats are used.
Table 1
Embodiment 2：The culture of Chinese hamster ovary celI (hereinafter referred to as cell)
Cell strain is CHO attached cell strains AC4.Initial medium uses the IMDM cell culture containing 10% fetal calf serum
Base (hereinafter referred to as blood serum medium), by cell with 2 × 105The quantity of cells/ml is inoculated in 6 porocyte culture plates, training
The condition of supporting is 37 DEG C, the incubator containing 5.0% carbon dioxide, and culture cell quantity reaches 2~6 × 106It is standby when cells/ml
With.
Embodiment 3：Handle the Chinese hamster ovary celI of culture
The cell that will be grown in blood serum medium after handling 1~5min using pancreatin, collects cell in the centrifugation of 15ml
Guan Zhong, 1000rpm centrifuge 3min, and the serum free medium 10ml washing cells for sucking preheating are primary, are finally settled to the nothing of 5ml
In blood serum medium, cell B measures the quantity of cell B using Trypan Blue staining cell B, spare.
Embodiment 4：The pretreatment of cell culture 6 orifice plates
Cell has a large amount of death during suspension, is deposited in order to which cell can be improved in serum free medium
Quantity living pre-processes 6 orifice plates using fiber link albumen (Fibronectin).Concrete operation step is as follows：Take pH value 7.0
~7.2 1 × PBS solution dilution fiber link albumen mother liquor (1mg/ml), obtains final working concentration 20~80 μ g/ml's
Mixed solution, then draw 1ml mixed solutions and be added to cell culture 6 orifice plates, processing time about 15~60min.
Embodiment 5：Cell suspension cultures
Using the serum free medium prepared in example 1, cell B is pressed 2 × 105The quantity of cells/ml is seeded to 6 holes
In plate, while control group is set, the growth conditions of observation cell B, while measuring the life of cell B after the micro somatomedin of addition
Long curve, to judge the growing state of cell B.Cell B is more apparent in the changing features in secondary stage, and cell B can be from adherent
The state of growth is gradually converted into the state of suspension growth.Growth conditions of the cell B in this stage are the key steps that cell suspends
Suddenly, time longer then cell B is easy death, and the time, too short unfavorable cell B suspended.The observing time of cell B is set as 3~5 days.
According to the state status of cell B growths, the cell B of suspension is forwarded to the 6 orifice plates equally handled, continues to cultivate.It can be normal
The cell B of growth will continue to retain, and finally screen suspension cell from the cell B of growth.
Embodiment 6：Western-Blotting detects the antibody expression after cell suspends
(1) SDS-PAG colloid concentrations are 10~15%, volume 10ml, and specific formula is that 100 μ are added in 10%SDS solution
10 μ l TEMED are added in l 10%APS, and concentration glue formula is that 40 μ l 10%APS are added in 4ml strike solution, finally
4 μ l TEMED are added；
(2) 20 μ l 5 × Loading Buffer, boiling water boiling 5min are added in sample preparation, 60 μ l cells and supernatants；
(3) point takes on 10 μ l to SD-PAGE colloids of sample, concentrates 80~90v of gel electrophoresis, electrophoresis 1h, detaches gel electrophoresis
120v, 1~2h of electrophoresis.Antibody protein is transferred on nitrocellulose membrane (PVDF), ice bath transferring film electric current 100mA, 1h；
(4) it measures 30ml 1 × TBST solution and 1.5g skimmed milk powers, mixing, 1~2h of close membrane is added；
(5) it measures 30ml 1 × TBST solution and 1.5g skimmed milk powers is added, detection antibody (the goat-anti people of 6 μ l is added in mixing
FITC-IgG, Sigma), 1~2h of label film, after clean 3 times；
(6) Western-Blotting developer solutions, exposure observation is added.
The embodiments of the present invention described above are not intended to limit the scope of the present invention.It is any in the present invention
Spirit and principle within made by modifications, equivalent substitutions and improvements etc., should be included in the claim protection model of the present invention
Within enclosing.
Claims (6)
1. expressing the Chinese hamster ovary celI serum free suspension cultural method of Rabies virus antibody, include the following steps：
(1) culture of cell strain：The cell strain is Chinese hamster ovary cell, and hereinafter referred to as cell；By cell with 2 ×
105~6 × 105The quantity of cells/ml is inoculated in blood serum medium, and condition of culture is 37 DEG C, the culture of 5.0% carbon dioxide
Case obtains cell A；It measures the growth curve of cell A and the growth curve of cell A after micro somatomedin is added；Institute
It is growth accelerator G-CSF-RCHP to state somatomedin, and is added to blood serum medium by 0.2~0.4 μ L/ml；
The blood serum medium is the IMDM liquid solutions containing 10% fetal calf serum；
(2) collection of cell：The cell A that will be grown in blood serum medium after handling 1~5min using pancreatin, collects cell A
In the centrifuge tube of 15ml, 1000rpm centrifuges 3min, and the 10ml free serum culture based sols for sucking 37 DEG C of preheatings wash cell one
Secondary, final constant volume obtains cell B, and use Trypan Blue dyeing counting cells in the serum free medium of volume 5ml
B measures the quantity of cell B；
The serum free medium is formulated by following components：
DMEM/F12 liquid solution 1ml, and filtration sterilization adjust pH to 7.0~7.2；
2~10ng/ml of Trace selenium；
Insulin 1-3 μ g/ml；
2~15 μ g/ml of transferrins；
Growth accelerator G-CSF-RCHP：0.2~0.4 μ l/ml；
Anti-cell nodule agent：0.1~0.5 μ l/ml；
And after the completion of the serum free medium is prepared, use preceding 37 DEG C of pre- stand-by heats；
(3) pretreatment of 6 orifice plates：It is 7.0~7.2 1 × BS solution to take pH value, and the fiber that diluted concentration is 1mg/ml links egg
In vain, it obtains final working concentration and is the mixed solution of 20~80 μ g/ml, then draw 1ml mixed solutions and 6 orifice plates are added, when processing
Between for 15~60min to get to pretreatment 6 orifice plates；
(4) cell culture：Cell B is pressed 2 × 105~6 × 105In the quantity inoculation pretreatment 6 orifice plates of cells/ml, then will be thin
Born of the same parents B successively in the IMDM fluid nutrient mediums containing 5% fetal calf serum, the IMDM fluid nutrient mediums containing 2.5% fetal calf serum, contain
Have it is foster by being commissioned to train in the IMDM fluid nutrient mediums of 1.25% fetal calf serum, the IMDM fluid nutrient mediums containing 0.1% fetal calf serum,
When cell B is full of adherent growth or after culture overnight growth, you can replace with what above-mentioned fetal calf serum ratio gradually reduced
IMDM fluid nutrient mediums, until replacing with serum free medium；While the cell B by micro- sem observation Jing Guo culture overnight
Form, and according to the state status of cell B growths, when cell B is gradually converted to suspension growth, and cellular morphology by adherent growth
Circle is converted to by arc or fusiformis, the not pretreated 6 orifice plates of cell B switchings of suspension are continued to cultivate, finally from growth
Cell B in screen suspension cell, and the suspension cell antibody expression filtered out is detected.
2. Chinese hamster ovary celI serum free suspension cultural method according to claim 1, it is characterised in that：In step (1), carefully
Born of the same parents are with 2 × 105The quantity of cells/ml is inoculated in blood serum medium.
3. Chinese hamster ovary celI serum free suspension cultural method according to claim 1, it is characterised in that：In step (4), cell B
By 2 × 105The quantity inoculation pretreatment 6 orifice plates of cells/ml.
4. Chinese hamster ovary celI serum free suspension cultural method according to claim 1, it is characterised in that：Cell B in step (4)
Incubation time be 3~5 days.
5. Chinese hamster ovary celI serum free suspension cultural method according to claim 1, it is characterised in that：In step (4), adopt
Cell B expression antibody is detected with the detection mode of Western-Blotting.
6. a kind of Chinese hamster ovary celI serum free medium of optimization, which is characterized in that composed of the following components：
DMEM/F12 liquid solutions：1ml, filtration sterilization, pH to 7.0~7.2；
Trace selenium：2~10ng/ml；
Insulin：1~3 μ g/ml；
Transferrins：2~15 μ g/ml；
Growth promotion dosage G-CSF-RCHP：0.2~0.4 μ l/ml；
Anti-cell nodule agent：0.1~0.5 μ l/ml.
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