CN105462934A - Additive used for increasing clone number of hybridoma cells and preparation method of additive - Google Patents
Additive used for increasing clone number of hybridoma cells and preparation method of additive Download PDFInfo
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- CN105462934A CN105462934A CN201610005108.9A CN201610005108A CN105462934A CN 105462934 A CN105462934 A CN 105462934A CN 201610005108 A CN201610005108 A CN 201610005108A CN 105462934 A CN105462934 A CN 105462934A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
- C12N5/12—Fused cells, e.g. hybridomas
- C12N5/16—Animal cells
- C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2306—Interleukin-6 (IL-6)
Abstract
The invention provides an additive used for increasing the clone number of hybridoma cells and a preparation method of the additive, and relates to an immunological research technology. The preparation method includes the following steps that 1, culture solution supernatant for eukaryotic cells capable of expressing IL-6 protein is acquired, wherein the cell culture solution supernatant contains expressed IL-6 protein; 2, the cell culture solution supernatant is subjected to ultrafiltration and filtration sterilization in sequence, and a cell culture solution supernatant concentrated solution is obtained; 3, sodium selenite is added into the cell culture solution supernatant concentrated solution, and the additive is obtained. As proved by experimental data, the clone number of the hybridoma cells can be increased by nearly 5 times after the additive is adopted. The invention further provides a hybridoma cell culture medium containing the additive.
Description
Technical field
The present invention relates to additive for improving hybridoma cell clone quantity and preparation method thereof, belonging to biological and field of medicaments.
Background technology
Immunology is current medical treatment and the requisite part of scientific research.After before 40 years, Edelman and Porter takes the lead in isolating immunoglobulin molecules, antibody has been applied to a lot of aspect.Antibody is applied to proteomics and diagnostics aspect to detect tumour and bacterial antigens; Can also synthesize using as vaccine and anti-tumor factor in Goat Milk and plant; Also can be used as pharmaceutical carrier instrument to treat virus, bacteriological infection and tumour.
The production of hybridoma technology and monoclonal antibody is that research work brings revolution, provides countless, unique monoclonal antibody reagent, for the qualification of specific antigens, separation, removing, activation or detection etc.The B cell hybridoma cell preparing secretory antibody depends on the high efficiency fusion of myeloma cell of antigen reactivity B cell and immortalization, and qualification subsequently with to can continued propagation can produce being separated of the clone of specific antibody.In the process of cytogamy, cell can be subject to damage in various degree.Obtaining optimal growth condition to make the hybridoma after fusion, increasing the output of hybridoma, in hybridoma cell cloneization is cultivated, need to add some extra somatomedin or fill-ins.Some laboratory can use cell feeder layer (splenocyte or peritoneal macrophage).Following several aspect is it is generally acknowledged in trophoblastic effect: 1, produce some cytokine to regulate the growth of hybridoma; 2, cell concn is increased to promote breeding (monoclonal antibody-hybridoma technology teaching materials, microbiology teaching and research room of The Fourth Military Medical University compiles) that is single or minority dispersion hybridoma Density dependence cell; 3, the meta-bolites that some is harmful is absorbed; 4, removing dead cell and fragment thereof is engulfed; 5, microbial growth (KeithJames, the eta.Humanmonoclonalantibodyproductioncurrentstatusandfut ureprospects.JImmunolMeth1987 of possible pollution is suppressed; 100:5) etc.Except feeder layer cells, the commercial additive (OrigenHCF.IGENInc, Gaithersburg, MD) that IL-6 originates also can be used.But when preparing feeder layer cells, each fusion all will kill 4-5 small white mouse, be unfavorable for animal welfare, and commercial hybridoma additive price costly.
Summary of the invention
The invention provides a kind of additive for improving hybridoma cell clone quantity, effectively can either promote the growth of hybridoma, can production cost be reduced again and watch for animals, meet the demand of scientific research and medical treatment.
Technical scheme provided by the invention is as follows:
For improving the preparation method of the additive of hybridoma clone quantity, comprise the steps:
(1) the eukaryotic nutrient solution supernatant can expressing IL-6 albumen is obtained, containing the IL-6 albumen of expressing in described cell culture supernatant;
(2) ultrafiltration is carried out to cell culture supernatant, then filtration sterilization, obtain cell culture supernatant concentrated solution;
(3) in cell culture supernatant concentrated solution, add Sodium Selenite, obtain described additive.
In technical scheme of the present invention, described ultrafiltration refers to, after carrying out ultrafiltration, then concentrates filtered solution with retaining low-molecular-weight super filter tube with the super filter tube retaining high molecular to cells and supernatant; Described filtration sterilization refers to, filters the cells and supernatant after concentrated with the filter membrane of 0.22 μm.
In technical schemes more of the present invention, described in retain the super filter tube of high molecular specification be selected from any one in 30KD, 50KD and 100KD, described in retain low-molecular-weight super filter tube specification be selected from any one in 3KD, 5KD and 10KD.Wherein one group is preferably: described in retain the specification 50KD of the super filter tube of high molecular, described in retain the specification 10KD of low-molecular-weight super filter tube.
In technical scheme of the present invention, the final concentration of preferred described Sodium Selenite is 1mg/L-10 μ g/L.
In technical scheme of the present invention, the eukaryotic cell preferably can expressing IL-6 albumen is selected from the Chinese hamster ovary celI system transforming and have the recombinant expression vector can expressing IL-6 albumen.Flp-In-293 in the further preferably Chinese hamster ovary celI system of described eukaryotic cell.
In technical scheme of the present invention, preferred described recombinant expression vector is pcDNA5/FRT-IL6, take pcDNA5/FRT as skeleton, pcDNA5/FRT and IL-6 full-length cDNA process KpnI and XbaI double digestion, through electrophoresis, cut after glue reclaims, spend the night with T4DNA ligase enzyme and connects, connection product conversion competent cell E.coliDH5a, the dull and stereotyped also picking list colony identification of coating Amp, identifies the plasmid called after pcDNA5/FRT-IL6 of correct positive colony.
In most of technical scheme of the present invention, described nutrient solution supernatant adopts the nutrient solution supernatant of eukaryotic cell early growth period, and described early growth period refers to that cultivation is no more than 48 hours.Eukaryotic cell can secrete a large amount of various somatomedin at early growth period, can grow fast by irritation cell, and these concentrated solutions join in hybridoma culture fluids as additive, can improve the surviving rate of hybridoma.
The additive for improving hybridoma cell clone quantity that the present invention also asks to protect the above-mentioned arbitrary preparation method's preparation of employing and obtains.
Additive provided by the invention may be used for the substratum making hybridoma, it is characterized in that, add described additive in containing the DMEM of 10% foetal calf serum, the volume ratio of the described additive of preferred interpolation is 1%-80%, more preferably 5%-75%, more preferably 5%-70%, more preferably 5%-65%, more preferably 5%-60%.As can be seen from experimental data of the present invention, add additive of the present invention optimum in about 20% effect, reduction ratio or raising ratio, its cultured cells clone number increasing amount is all successively decreased, but can find out that adding proportion arbitrary numerical value between 1-80% can play the effect improving clone's number.
The present invention is based on above-mentioned additive, also the method for hybridoma is cultivated in request protection, it is characterized in that, in its substratum, adds above-mentioned additive, or adopt the substratum of described hybridoma.
The present invention for improving the additive of hybridoma clone quantity, with artificial culture containing the cell culture supernatant of IL-6 albumen for after raw material ultrafiltration and concentration, after adding Sodium Selenite and get final product.Can be used as hybridoma somatomedin to use.Additive provided by the invention is except traditional cell additive such as IL-6 and Sodium Selenite is contained in the inside, containing the various somatomedins also having the secretion of eukaryotic cell process of growth, can grow fast by irritation cell, these concentrated solutions join in hybridoma culture fluids as additive, can improve the surviving rate of hybridoma, preparation method is simple to operate, cost value is cheap.Sodium Selenite Wheat Protein, improves growth velocity and activity.
Improve compared with the method for hybridoma quantity with traditional by feeder layer cells; the present invention adopts vitro culture can express the eukaryotic cell of IL-6 to prepare additive; the additive that effectively can improve hybridoma quantity can be obtained without the need to murdering mouse; and experiment proves; the effect of additive raising hybridoma quantity of the present invention can reach more than 2 times of feeder layer cells; not only save cost and time prepared by antibody, also protect animal.Compared with commercial hybridoma additive, addictive preparation method of the present invention is simple, the reagent used and consumables cost low, greatly reduce Financial cost and human cost, be particularly useful for large-scale antibody producing.
Accompanying drawing explanation
The expression of Fig. 1 .IL-6 in cells and supernatant and cracking supernatant.
Embodiment
Below with reference to specific embodiment, the present invention will be described, it should be noted that, these embodiments only illustratively and illustrate, and can not be interpreted as limitation of the present invention.
The biomaterial (microorganism, Flp-In-293 cell, expression vector) that the present invention uses and all commercially available acquisition of reagent, also there is preservation in our unit, can ensure to provide for proof test to the public from the applying date.
Flp-In-293 belongs to Chinese hamster ovary celI system.
Embodiment 1, for improving the preparation of additive of hybridoma clone quantity
1, the Design and synthesis of primer
According to known Musmusculusinterleukin6 (Il6) sequence (GeNBank GeneID:16193), by JavaCodonAdaptationTool software optimization sequence, obtain sequence IL-6* (as shown in SEQIDNO.:1).This sequence is synthesized by the raw work in Shanghai and is cloned on pGH plasmid, obtains pGH-IL-6* plasmid.Its PCR primer is:
Upstream primer IL-F:5 '-AAGGTACCatgaaattcctgtctgctcg-3 ',
Downstream primer IL-R:5 '-TATCTAGAttaggtctgacgggtag-3 '
Upstream primer and downstream primer respectively with KpnI, XbaI enzyme cutting site.This primer is synthesized by the raw work in Shanghai.
2, carrier for expression of eukaryon pcDNA5/FRT-IL6 is built
With pGH-IL-6* plasmid for template, pcr amplification obtains IL-6* sequence, with KpnI, after XbaI restriction enzyme carries out double digestion to this sequence, agarose gel electrophoresis reclaims object fragment, use KpnI, XbaI double digestion carrier for expression of eukaryon pcDNA5/FRT (purchased from Invitrogen company) simultaneously.Object fragment and carrier be connected at 16 DEG C and spend the night, 42 DEG C of heat-shock transformed competent cell E.coliDH5 α, coat on Amp flat board and cultivate.Picking list bacterium colony, use carrier universal primer M13R/M13F (M13F:GTAAAACGACGGCCAGTM13R:AACAGCTATGACCATG) to carry out single bacterium colony PCR and identify, obtain positive colony, the size of its PCR primer is consistent with expection; Extract the plasmid of this positive colony simultaneously, carry out double digestion with KpnI, XbaI, the size of digestion products is consistent with expection, obtains carrier for expression of eukaryon thus, called after pcDNA5/FRT-IL6.
3, carrier for expression of eukaryon pcDNA5/FRT-IL6 transfection Flp-In-293 cell
24h before transfection, digests Flp-In-293 cell (purchased from Invitrogen company) and is inoculated in 6 orifice plates, 5 × 10
5cells/well.Before transfection by Opti-MEM substratum, without blood nonreactive DMEM cultivation based on 37 DEG C of water-bath preheatings, plasmid, Lipofectamine (purchased from Invitrogen company) are carried out ice bath; Add 1 μ g recombinant plasmid pcDNA5/FRT-IL6 to the 1.5ml finger-type Guan Zhongzhi cumulative volume 100 μ l containing Opti-MEM substratum, concussion mixing; Stirring-type adds 6uLLipofectamine in the 2ml finger-type pipe containing 94 μ lOpti-MEM substratum; Being stirred by plasmid/Opti-MEM respectively adds in Lipofectamine/Opti-MEM, and after leaving standstill 25min, stirring-type adds 0.6mlOpti-MEM; Transfection reagent being added in advance with cleaning without blood nonreactive DMEM in the cell gone over, being placed in 37 DEG C, the incubator 5h of 5%CO2; Suck culture supernatant, every hole adds 2ml perfect medium (containing 100 μ g/mlZeocin), is placed in incubator and continues to cultivate; Peptic cell after transfection 48h, in some flat boards, carry out resistance screening with containing after the perfect medium dilution of 120 μ g/mlHygromycinB, every 4-5 days changes liquid once, has the macroscopic clone that individual cells grows up to after 3-4 week.Treat that clone grows to suitable size, suck selective medium, draw a small amount of pancreas enzyme-EDTA point on individual cells clone, pause and digest for several seconds, when cell mass comes off from flat board, suck back Digestive system, transfer in the selective medium added in advance and stop digestion, enlarged culturing.Take limiting dilution assay to carry out subclone 2 times to the cell of enlarged culturing, obtain cell strain called after 2G6.
4, expression product detects
Use pancreatin-EDTA difference digestive inoculation Flp-In-293 cell and 2G6 cell in 6 orifice plates, 5 × 10
5cells/well; Perfect medium (DMEM+10% foetal calf serum) is adopted to cultivate, 37 DEG C, 48h.Collecting cell supernatant; Use trypsin digestion cell, add appropriate PBS by cell suspension and carry out multigelation cracking; Double crush syndrome method is used to detect the expression of recombinant vectors pcDNA5/FRT-IL6 the cell conditioned medium collected, cracking supernatant.
Use coating buffer (carbonate buffer solution, pH9.6) to dilute Sheet to resist, 100 μ l/ holes, put 4 DEG C and spend the night; Wash (washings: PBS, pH7.4 containing 0.05%Tween-20) according to a conventional method and close (confining liquid: the washings containing 1%BSA); Add cells and supernatant, 100 μ l/ holes, hatch 2h for 37 DEG C; Wash 3 times, add the polyclonal antibody of the anti-IL6 of HRP mark, 100 μ l/ holes, hatch 1h for 37 DEG C; Wash 3 times, add substrate nitrite ion, lucifuge reaction 15min, add stop buffer 50 μ l/ hole, read absorbancy (OD450), experimental result is as Fig. 1.As can be seen from the figure IL-6 mainly expresses in the supernatant liquor cultivated.
5, the preparation of Growth of Hybridoma Cell supplement
Inoculate 2G6 cell in 6 orifice plates, 5 × 10
5cells/well; Perfect medium (DMEM+10% foetal calf serum) is adopted to cultivate, 37 DEG C, 48h.Collecting cell supernatant.Enchylema supernatant is with after the super filter tube ultrafiltration retaining 50KD, and the filtered solution super filter tube retaining 10KD concentrates, and then concentrated solution is through 0.22 μm of sterile filtration.100 μ g/L Sodium Selenites are added, as final additive in filtered liquid.
Embodiment 2, for improving the effect measuring of additive of hybridoma clone quantity
1, the preparation of hybridoma
(1) animal immune
The 20mM phosphate buffered saline buffer (pH7.4) using 100 μ l to contain 0.15MNaCL dilutes 50 μ gBSA albumen, is mixed and made into emulsion with the Freund's complete adjuvant of equivalent.8-12 week BALA the subcutaneous injection of C mouse multiple spot.Equivalent amount of antigen and Freund's incomplete adjuvant booster immunization is got once after 3 weeks; 4 weeks, interval again, gets and does not add adjuvant through vein supplementary immunization with amount antigen.
(2) preparation of immune spleen cell suspension
After final immunization the 3rd day, put to death immune mouse.In Bechtop, win spleen be placed on 100 object stainless steel sifts on the net, mill gently with piston, and rinse with serum free medium, collecting cell.Wash 3 times with DMEM substratum, be suspended in 10MLDMEM substratum, count for subsequent use.
(3) cytogamy
The SP2/0 myeloma cell 2 × 10 taken the logarithm vegetative period
7immune spleen cell 2 × 10 prepared by individual and step (2)
8individual, add centrifuge tube mixing.By two kinds of cells with 200 × g centrifugal 10 minutes of mixing, cytogamy is carried out with PEG1450 after removing supernatant, by cell 2 times of HAT substratum (the DMEM substratum containing xanthoglobulin, aminopterin, thymidine, 10% foetal calf serum) dilution of merging, adjustment cell concn to 1 × 10
5individual/ml.
2, the preparation of feeder cell
Get BALA C mouse, sacrificed by exsanguination.In super clean bench, cut off skin of chest, expose belly, get 5ml serum free medium and inject abdominal cavity, soft 1-2 minute, then by cell suspension sucking-off, move in centrifuge tube.Serum free medium washs 1 time, is suspended in the DMEM substratum of 10% foetal calf serum, adjustment concentration to 2 × 10
5individual/ml, is inoculated in 96 well culture plates, and every hole 0.1ml is for subsequent use.
3, the preparation of Growth of Hybridoma Cell supplement
Inoculate 2G6 cell in 6 orifice plates, 5 × 10
5cells/well; Perfect medium (DMEM+10% foetal calf serum) is adopted to cultivate, 37 DEG C, 48h.Collecting cell supernatant.Enchylema supernatant is with after the super filter tube ultrafiltration retaining 50KD, and the filtered solution super filter tube retaining 10KD concentrates, and then concentrated solution is through 0.22 μm of sterile filtration.100 μ g/L Sodium Selenites are added, as final additive in filtered liquid.Get 96 porocyte culture plates, every plate adds additive 0 μ l/ hole respectively, 5 μ l/ holes, 10 μ l/ holes, 20 μ l/ holes, 40 μ l/ holes and 80 μ l/ holes.Finally use the DMEM culture medium supplemented of 10% foetal calf serum to 100 μ l, do 3 repetitions.
4, additive and feeder cell contrast the promoter action of Growth of Hybridoma Cell
Fused cell liquid step 1 prepared by every hole 100 μ l joins containing in the culture plate of feeder cell or growth supplement of preparation in advance, in 37 DEG C, cultivates 2-3 week in 5%CO2 incubator, observes the growing state of hybridoma clone in 96 orifice plates.
Result is as follows:
Table 1 additive and feeder cell contrast the promoter action of Growth of Hybridoma Cell
As can be seen from the table, add feeder cell and all effectively can improve the quantity of hybridoma by growth supplement prepared by above-mentioned steps.Add the quantity that feeder cell can improve 170% hybridoma clone, and add the quantity that growth supplement can improve hybridoma clone more significantly.
The growth supplement that every hole adds 5 μ l improve 251%;
The growth supplement that every hole adds 10 μ l improve 292%;
The growth supplement that every hole adds 20 μ l improve 489%;
The growth supplement that every hole adds 40 μ l improve 200%;
The growth supplement that every hole adds 80 μ l improve 10%.
Result proves, the Growth of Hybridoma Cell supplement prepared by the inventive method can improve the quantity of hybridoma clone effectively, and then improves the probability filtering out positive monoclonal antibody strain, the time a large amount of for the saving of preparation monoclonal antibody and cost.
Claims (10)
1., for improving the preparation method of the additive of hybridoma clone quantity, comprise the steps:
(1) the eukaryotic nutrient solution supernatant can expressing IL-6 albumen is obtained, containing the IL-6 albumen of expressing in described cell culture supernatant;
(2) ultrafiltration is carried out to cell culture supernatant, then filtration sterilization, obtain cell culture supernatant concentrated solution;
(3) in cell culture supernatant concentrated solution, add Sodium Selenite, obtain described additive.
2. preparation method according to claim 1, is characterized in that, described ultrafiltration refers to, after carrying out ultrafiltration, then concentrates filtered solution with retaining low-molecular-weight super filter tube with the super filter tube retaining high molecular to cells and supernatant; Described filtration sterilization refers to, filters the cells and supernatant after concentrated with the filter membrane of 0.22 μm.
3. preparation method according to claim 2, it is characterized in that, the described specification retaining the super filter tube of high molecular is selected from any one in 30KD, 50KD and 100KD, described in retain low-molecular-weight super filter tube specification be selected from any one in 3KD, 5KD and 10KD.
4. preparation method according to claim 1, is characterized in that, the final concentration of described Sodium Selenite is 1mg/L-10 μ g/L.
5. preparation method according to claim 1, the eukaryotic cell can expressing IL-6 albumen is selected from the Chinese hamster ovary celI system transforming and have the recombinant expression vector can expressing IL-6 albumen.
6. preparation method according to claim 5, described recombinant expression vector is pcDNA5/FRT-IL6, take pcDNA5/FRT as skeleton, pcDNA5/FRT and IL-6 full-length cDNA process KpnI and XbaI double digestion, through electrophoresis, cut after glue reclaims, spend the night with T4DNA ligase enzyme and connects, connection product conversion competent cell E.coliDH5a, the dull and stereotyped also picking list colony identification of coating Amp, identifies the plasmid called after pcDNA5/FRT-IL6 of correct positive colony.
7. the arbitrary described preparation method of claim 1-6, described nutrient solution supernatant is that eukaryotic cell cultivates the nutrient solution supernatant being no more than 48 hours.
8. for improving the additive of hybridoma cell clone quantity, it is characterized in that, adopting the arbitrary described preparation method preparation of claim 1-7.
9. the substratum of hybridoma, it is characterized in that, be added with arbitrary additive according to claim 8 in containing the DMEM of 10% foetal calf serum, the volume ratio of the described additive added is 1-80%, more preferably 5%-75%, more preferably 5%-70%, more preferably 5%-65%, more preferably 5%-60%.
10. cultivate the method for hybridoma, it is characterized in that, in its substratum, add additive according to claim 8, or adopt substratum according to claim 9.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108624622A (en) * | 2018-05-16 | 2018-10-09 | 湖南艾佳生物科技股份有限公司 | A kind of genetically engineered cell strain that can secrete mouse interleukin -6 based on CRISPR-Cas9 systems structure |
| CN113493774A (en) * | 2020-03-20 | 2021-10-12 | 南京松天盛科生物科技有限公司 | Culture medium for improving hybridoma cell fusion rate |
| CN113755449A (en) * | 2021-09-30 | 2021-12-07 | 南京天纵易康生物科技股份有限公司 | Nutritional supplement for improving survival rate of hybridoma cells, culture medium and culture method |
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| CN103898041A (en) * | 2012-12-25 | 2014-07-02 | 深圳先进技术研究院 | Culture method of hybridomas |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108624622A (en) * | 2018-05-16 | 2018-10-09 | 湖南艾佳生物科技股份有限公司 | A kind of genetically engineered cell strain that can secrete mouse interleukin -6 based on CRISPR-Cas9 systems structure |
| CN113493774A (en) * | 2020-03-20 | 2021-10-12 | 南京松天盛科生物科技有限公司 | Culture medium for improving hybridoma cell fusion rate |
| CN113493774B (en) * | 2020-03-20 | 2024-01-16 | 南京松天盛科生物科技有限公司 | Culture medium for improving hybridoma cell fusion rate |
| CN113755449A (en) * | 2021-09-30 | 2021-12-07 | 南京天纵易康生物科技股份有限公司 | Nutritional supplement for improving survival rate of hybridoma cells, culture medium and culture method |
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