CN105462934B - For improving the additive and preparation method thereof of hybridoma cell clone quantity - Google Patents
For improving the additive and preparation method thereof of hybridoma cell clone quantity Download PDFInfo
- Publication number
- CN105462934B CN105462934B CN201610005108.9A CN201610005108A CN105462934B CN 105462934 B CN105462934 B CN 105462934B CN 201610005108 A CN201610005108 A CN 201610005108A CN 105462934 B CN105462934 B CN 105462934B
- Authority
- CN
- China
- Prior art keywords
- additive
- cell
- hybridoma
- preparation
- cell culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- 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
-
- 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)
Landscapes
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention is used to improve the additive and preparation method thereof of hybridoma cell clone quantity, it is related to immunological investigation technology, include the following steps: that (1) obtains the culture solution supernatant that can express the eukaryocyte of IL-6 albumen, the IL-6 albumen containing expression in the cell culture supernatant；(2) ultrafiltration is carried out to cell culture supernatant, then filtration sterilization obtains cell culture supernatant concentrate；(3) sodium selenite is added into cell culture supernatant concentrate, obtains the additive.Test data proves, can be improved nearly 5 times using hybridoma cell clone quantity after the additive.The present invention still further provides the Hybridoma Cell Culture base containing above-mentioned additive.
Description
Technical field
The present invention relates to the additives and preparation method thereof for improving hybridoma cell clone quantity, belong to biology and doctor
Medicine field.
Background technique
Immunology is current medical treatment and the essential a part of scientific research.It takes the lead in point from Edelman and Porter before 40 years
After separating out immunoglobulin molecules, antibody has been applied to many aspects.Antibody is applied to proteomics and diagnostics side
Face is to detect tumour and bacterial antigens；It can also be synthesized in Goat Milk and plant using as vaccine and anti-tumor factor；May be used also
As pharmaceutical carrier tool to treat virus, bacterium infection and tumour.
The research work that is produced as of hybridoma technology and monoclonal antibody brings revolution, provides countless, unique
Monoclonal antibody reagent, for the identification of specific antigen, separation, removing, activation or detection etc..The B for preparing secretory antibody is thin
Born of the same parents' hybridoma dependent on antigen reactivity B cell with the myeloma cell of immortalization is efficient merges, and it is subsequent
The separation of identification and the cell line that can generate specific antibody to energy continued propagation.During cell fusion, cell meeting
By different degrees of damage.In order to make fused hybridoma obtain the yield of the best growing condition, increase hybridoma,
In hybridoma cell clone culture, need to add some additional growth factors or supplement.Some laboratories will use
Cell feeder layer (splenocyte or peritoneal macrophage).Trophoblastic effect is it is generally acknowledged that have following aspects: 1, generating certain
A little cell factors adjust the growth of hybridoma；2, increase cell concentration to promote single or a small number of dispersion hybridoma density
The breeding of dependent cells (monoclonal antibody-hybridoma technology handout, microbiology teaching and research room, The Fourth Military Medical University compile)；3, it absorbs
Certain harmful metabolites；4, dead cell and its fragment are removed in phagocytosis；5, inhibit the growth of the microorganism of possible pollution
(Keith James, et a.Human monoclonal antibody production current status and
future prospects.J Immunol Meth 1987；100:5) etc..In addition to feeder cells, IL-6 also can be used
The additive (Origen HCF.IGEN Inc, Gaithersburg, MD) of the commercialization in source.However, preparing feeder cells
When, fusion will kill 4-5 small white mouse every time, it is unfavorable for animal welfare, and the hybridoma additive price being commercialized compares
Costly.
Summary of the invention
The present invention provides a kind of for improving the additive of hybridoma cell clone quantity, can either effectively facilitate hybridoma
The growth of cell, and production cost can be reduced and protect animal, meet the needs of scientific research and medical treatment.
Technical solution provided by the invention is as follows:
For improving the preparation method of the additive of hybridoma clone quantity, include the following steps:
(1) the culture solution supernatant that can express the eukaryocyte of IL-6 albumen is obtained, is contained in the cell culture supernatant
The IL-6 albumen of expression；
(2) ultrafiltration is carried out to cell culture supernatant, then filtration sterilization obtains cell culture supernatant concentrate；
(3) sodium selenite is added into cell culture supernatant concentrate, obtains the additive.
In technical solution of the present invention, the ultrafiltration refers to, with the super filter tube of retention high molecular weight to cells and supernatant
Carry out ultrafiltration after, then with retain low molecular weight super filter tube filtered solution is concentrated；The filtration sterilization refers to, with 0.22 μm
Filter membrane the cells and supernatant after concentration is filtered.
In some technical solutions of the invention, it is described retention high molecular weight super filter tube specification be selected from 30KD, 50KD and
Any one of 100KD, it is described retention low molecular weight super filter tube specification in 3KD, 5KD and 10KD any one
Kind.Wherein one group it is preferred are as follows: the specification 50KD of the super filter tube of the retention high molecular weight, the super filter tube of the retention low molecular weight
Specification 10KD.
In technical solution of the present invention, the final concentration of 1mg/L-10 μ g/L of the preferably described sodium selenite.
In technical solution of the present invention, the eukaryocyte that can preferably express IL-6 albumen, which is selected to convert to have, can express IL-6 egg
The CHO cell line of white recombinant expression carrier.Flp-In-293 in the further preferred CHO cell line of eukaryocyte.
In technical solution of the present invention, it is with pcDNA5/ that the preferably described recombinant expression carrier, which is pcDNA5/FRT-IL6,
FRT is skeleton, pcDNA5/FRT and IL-6 full-length cDNA, which passes through, uses KpnI and XbaI double digestion, after electrophoresis, gel extraction,
It is connected overnight with T4DNA ligase, connection product transformed competence colibacillus cell E.coli DH5a, coating Amp plate and picking single bacterium
Identification is fallen, identifies that the plasmid of correct positive colony is named as pcDNA5/FRT-IL6.
In most of technical solutions of the invention, the culture solution supernatant uses the culture solution of eukaryocyte early growth period
Supernatant, the early growth period refer to that culture is no more than 48 hours.Eukaryocyte can secrete a large amount of various growth factors in early growth period,
Cell fast-growth can be stimulated, these concentrates are added in hybridoma culture fluids as additive, and hybridoma can be improved
The survival rate of cell.
The present invention is also claimed to be used to improve hybridoma using what is be prepared using any of the above-described preparation method
Clone the additive of quantity.
Additive provided by the invention can be used for being made the culture medium of hybridoma, which is characterized in that be to contain
The additive is added in the DMEM of 10% fetal calf serum, the volume ratio of the additive preferably added is 1%-
80%, further preferably 5%-75%, further preferably 5%-70%, further preferably 5%-65%, it is further excellent
It is selected as 5%-60%.From experimental data of the invention can be seen that addition additive of the present invention it is optimal in 20% or so effect, drop
Low ratio improves ratio, and the number of cell clones raising amount of culture is all successively decreased, but it can be seen that adding proportion exists
Any value can play the role of improving clone's number between 1-80%.
The present invention is based on above-mentioned additive, the method that culture hybridoma is also claimed, which is characterized in that in its training
It supports and adds above-mentioned additive in base, or the culture medium using the hybridoma.
The present invention is used to improve the additive of hybridoma clone quantity, with the cell training containing IL-6 albumen manually cultivated
Nutrient solution supernatant is after raw material is concentrated by ultrafiltration, to be added after sodium selenite to obtain the final product.It can be used as the use of hybridoma somatomedin.The present invention
The additive of offer is other than the cell additive traditional containing IL-6 and sodium selenite etc. of the inside, containing there are also eukaryocytes to grow
The various growth factors of process secretion, can stimulate cell fast-growth, these concentrates are added to hybridoma as additive
In culture solution, the survival rate of hybridoma can be improved, preparation method is easy to operate, cost price is cheap.Sodium selenite has
Antioxidation improves growth rate and activity.
Compared with traditional method for improving hybridoma quantity by feeder cells, the present invention uses in vitro culture
The eukaryocyte of IL-6 can be expressed to prepare additive, can be obtained without murdering mouse can effectively improve hybridoma
The additive of quantity, and it is demonstrated experimentally that the effect that additive of the invention improves hybridoma quantity can achieve raising
The 2 times or more of confluent monolayer cells has not only saved cost and the time of Antibody preparation, has also protected animal.With the hybridoma of commercialization
Additive is compared, and addictive preparation method of the invention is simple and easy, and used reagent and consumables cost are low, is greatly reduced
Economic cost and human cost are particularly suitable for large-scale antibody producing.
Detailed description of the invention
Expression of Fig. 1 .IL-6 in cells and supernatant and cracking supernatant.
Specific embodiment
Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only as explanation
And explanation, and be not considered as limiting the invention.
Biomaterial (microorganism, Flp-In-293 cell, expression vector) that the present invention uses and reagent is commercially available obtains
, also there is preservation in our unit, it is ensured that provides from the applying date to the public and is used for verification test.
Flp-In-293 belongs to CHO cell line.
The preparation of embodiment 1, additive for improving hybridoma clone quantity
1, the design and synthesis of primer
According to known Mus musculus interleukin 6 (Il6) sequence (GeNBank GeneID:16193),
With Java Codon Adaptation Tool software optimization sequence, obtain sequence IL-6* (as shown in SEQ ID NO.:1).It should
Sequence is synthesized by the raw work in Shanghai and is cloned on pGH plasmid, obtains pGH-IL-6* plasmid.Its PCR primer are as follows:
Upstream primer IL-F:5 '-AAGGTACCatgaaattcctgtctgctcg-3 ',
Downstream primer IL-R:5 '-TATCTAGAttaggtctgacgggtag-3 '
Upstream primer and downstream primer are respectively provided with KpnI, XbaI enzyme cutting site.The primer is synthesized by the raw work in Shanghai.
2, carrier for expression of eukaryon pcDNA5/FRT-IL6 is constructed
Using pGH-IL-6* plasmid as template, PCR amplification obtains IL-6* sequence, with KpnI, XbaI restriction enzyme pair
The sequence carry out double digestion after, agarose gel electrophoresis recycle target fragment, while use KpnI, XbaI double digestion eukaryotic expression load
Body pcDNA5/FRT (is purchased from Invitrogen company).Target fragment and carrier are stayed overnight in 16 DEG C of connections, 42 DEG C heat-shock transformed
Competent cell E.coli DH5 α, is coated on Amp plate and cultivates.Picking single colonie uses carrier universal primer M13R/
M13F (M13F:GTAAAACGACGGCCAGT M13R:AACAGCTATGACCATG) carries out single colonie PCR identification, obtains positive
The size of clone, PCR product are consistent with expection；The plasmid for extracting the positive colony simultaneously carries out double enzymes with KpnI, XbaI
It cuts, the size of digestion products is consistent with expection, thus to obtain carrier for expression of eukaryon, is named as pcDNA5/FRT-IL6.
3, carrier for expression of eukaryon pcDNA5/FRT-IL6 transfects Flp-In-293 cell
Before transfection for 24 hours, Flp-In-293 cell (being purchased from Invitrogen company) is digested and is inoculated in 6 orifice plates
In, 5 × 105Cells/well.It is preheated by Opti-MEM culture medium, without blood nonreactive DMEM culture based on 37 DEG C of water-baths before transfection, by matter
Grain, Lipofectamine (being purchased from Invitrogen company) carry out ice bath；1 μ g recombinant plasmid pcDNA5/FRT-IL6 is added extremely
100 μ l of 1.5ml finger-type Guan Zhongzhi total volume containing Opti-MEM culture medium, concussion mix；6uL is added in stirring-type
Lipofectamine is into the 2ml finger-type pipe containing 94 μ l Opti-MEM culture mediums；Plasmid/Opti-MEM stirring is added respectively
Enter in Lipofectamine/Opti-MEM, 0.6ml Opti-MEM is added in stirring-type after standing 25min；Transfection reagent is added
It is cleaned in the cell gone over no blood nonreactive DMEM in advance, is placed in 37 DEG C, 5h in the incubator of 5%CO2；It sucks in culture
Clearly, every hole adds 2ml complete medium (containing 100 μ g/ml Zeocin), is placed in and continues to cultivate in incubator；It is digested after transfection 48h
Cell carries out resistance screening, every 4- after being diluted with the complete medium containing 120 μ g/ml Hygromycin B in several plates
It changes the liquid once within 5 days, the macroscopic clone that individual cells grow up to is had after 3-4 weeks.Suitable size is grown to wait clone, is sucked
Selective medium draws a small amount of pancreas enzyme -EDTA point on individual cells clone, pauses and digested for several seconds, to cell mass from
Digestive juice is sucked back when falling off on plate, is transferred in the selective medium being previously added and terminates digestion, expands culture.To expansion
The cell of culture takes limiting dilution assay to carry out subclone 2 times, obtains cell strain and is named as 2G6.
4, expression product detects
Using pancreatin-EDTA difference digestive inoculation Flp-In-293 cell and 2G6 cell in 6 orifice plates, 5 × 105Carefully
Born of the same parents/hole；It is cultivated using complete medium (DMEM+10% fetal calf serum), 37 DEG C, 48h.Collect cell conditioned medium；Disappeared using pancreatin
Change cell, appropriate PBS is added, cell suspends and carries out multigelation cracking；The cell conditioned medium of collection, cracking supernatant are used
The expression of double crush syndrome method detection recombinant vector pcDNA5/FRT-IL6.
Coating monoclonal antibody is diluted using coating buffer (carbonate buffer solution, pH9.6), 100 holes μ l/ set 4 DEG C overnight；Routinely
Method washs (cleaning solution: the PBS containing 0.05%Tween-20, pH7.4) and closing (confining liquid: the cleaning solution containing 1%BSA)；
Cells and supernatant, 100 holes μ l/, 37 DEG C of incubation 2h are added；The polyclonal antibody of the anti-IL6 of HRP label is added in washing 3 times,
100 holes μ l/, 37 DEG C of incubation 1h；Substrate developing solution is added in washing 3 times, is protected from light 15min, and 50 hole μ l/ of terminate liquid is added, and reads
It takes absorbance (OD450), experimental result such as Fig. 1.As can be seen from the figure IL-6 is mainly expressed in the supernatant of culture.
5, the preparation of Growth of Hybridoma Cell replenishers
2G6 cell is inoculated in 6 orifice plates, 5 × 105Cells/well；Using complete medium (DMEM+10% fetal calf serum)
Culture, 37 DEG C, 48h.Collect cell conditioned medium.After super filter tube ultrafiltration of the cell liquid supernatant with retention 50KD, filtered solution retention
The super filter tube of 10KD is concentrated, and then concentrate is sterile filtered through 0.22 μm.100 μ g/L sodium selenites are added in filtered fluid, as
Final additive.
The effect measuring of embodiment 2, additive for improving hybridoma clone quantity
1, the preparation of hybridoma
(1) animal immune
50 μ g BSA albumen are diluted using the 20mM phosphate buffer (pH7.4) that 100 μ l contain 0.15M NaCL, with etc.
The Freund's complete adjuvant of amount is mixed and made into emulsion.8-12 weeks BALA C mice multiple spot subcutaneous injection.Take equivalent anti-after 3 weeks
Former and Freund's incomplete adjuvant booster immunization is primary；It is spaced again 4 weeks, takes same amount antigen that adjuvant is not added through vein supplementary immunization.
(2) preparation of spleen cell suspension is immunized
The 3rd day after final immunization, immune mouse is put to death.Spleen is won in superclean bench, and to be placed on 100 purposes stainless
It on steel sieve, is gently milled with piston, and rinsed with serum free medium, collects cell.3 are washed with DMEM culture medium
It is secondary, it is suspended in 10ML DMEM culture medium, counts spare.
(3) cell fusion
The SP2/0 myeloma cell 2 × 10 of logarithmic growth phase7The immune spleen cell 2 × 10 of a and step (2) preparation8
It is a, centrifuge tube mixing is added.By two kinds of cells of mixing with 200 × g centrifugation 10 minutes, carried out after removing supernatant with PEG1450
2 times of HAT culture mediums of the cell of fusion (are contained hypoxanthine, aminopterin, thymidine, 10% fetal calf serum by cell fusion
DMEM culture medium) dilution, adjustment cell concentration to 1 × 105A/ml.
2, the preparation of feeder cells
Take BALA C mice, sacrificed by exsanguination.Skin of chest is cut off in super-clean bench, exposure abdomen takes 5ml free serum culture
Base injects abdominal cavity, 1-2 minutes soft, and then cell suspension is sucked out, and moves into centrifuge tube.Serum free medium washs 1 time, hangs
Float in the DMEM culture medium of 10% fetal calf serum, adjustment concentration to 2 × 105A/ml is inoculated in 96 well culture plates, every hole
0.1ml, it is spare.
3, the preparation of Growth of Hybridoma Cell replenishers
2G6 cell is inoculated in 6 orifice plates, 5 × 105Cells/well；Using complete medium (DMEM+10% fetal calf serum)
Culture, 37 DEG C, 48h.Collect cell conditioned medium.After super filter tube ultrafiltration of the cell liquid supernatant with retention 50KD, filtered solution retention
The super filter tube of 10KD is concentrated, and then concentrate is sterile filtered through 0.22 μm.100 μ g/L sodium selenites are added in filtered fluid, as
Final additive.96 porocyte culture plates are taken, every plate is separately added into 0 hole μ l/ of additive, 5 holes μ l/, 10 holes μ l/, 20 μ l/
Hole, 40 holes μ l/ and 80 holes μ l/.100 μ l finally are added to the DMEM culture medium of 10% fetal calf serum, do 3 repetitions.
4, additive and feeder cells compare the facilitation of Growth of Hybridoma Cell
Fused cell liquid prepared by step 1 is added to by every 100 μ l of hole prepare in advance containing feeder cells or growth
It in the culture plate of replenishers, is cultivated 2-3 weeks in 37 DEG C, 5%CO2 incubator, observes the growth of hybridoma clone in 96 orifice plates
Situation.
As a result as follows:
1 additive of table and feeder cells compare the facilitation of Growth of Hybridoma Cell
As can be seen from the table, feeder cells are added and can effectively be mentioned by growth replenishers prepared by above-mentioned steps
The quantity of high hybridoma.The quantity of 170% hybridoma clone can be improved in addition feeder cells, and adds growth replenishers
The quantity of hybridoma clone can be improved more significantly.
The growth replenishers that 5 μ l are added in every hole improve 251%；
The growth replenishers that 10 μ l are added in every hole improve 292%；
The growth replenishers that 20 μ l are added in every hole improve 489%；
The growth replenishers that 40 μ l are added in every hole improve 200%；
The growth replenishers that 80 μ l are added in every hole improve 10%.
The results show that hybridoma gram can be effectively improved by Growth of Hybridoma Cell replenishers prepared by the method for the present invention
Grand quantity, and then the probability for filtering out positive monoclonal antibody strain is improved, a large amount of time is saved for preparation monoclonal antibody
And cost.
Claims (6)
1. the preparation method for the additive for improving hybridoma clone quantity, includes the following steps:
(1) the culture solution supernatant within obtaining eukaryocyte 48 hours that can express IL-6 albumen, the cell culture supernatant
In containing expression IL-6 albumen；
(2) with retention 50KD super filter tube ultrafiltration is carried out to cell culture supernatant after, then with retain 10KD super filter tube to filter
It crosses liquid to be concentrated, then filtration sterilization, obtains cell culture supernatant concentrate；
(3) sodium selenite is added into cell culture supernatant concentrate to final concentration of 1mg/L-10 μ g/L, adds described in acquisition
Add agent；
The eukaryocyte for expressing IL-6 albumen, which is selected from conversion, has the CHO for the recombinant expression carrier that can express IL-6 albumen thin
Born of the same parents system.
2. preparation method according to claim 1, which is characterized in that the filtration sterilization refers to, with 0.22 μm of filter membrane
Cell culture supernatant after concentration is filtered.
3. preparation method according to claim 1, the recombinant expression carrier is pcDNA5/FRT-IL6, be with
PcDNA5/FRT is skeleton, pcDNA5/FRT and IL-6 full-length cDNA, which passes through, uses KpnI and XbaI double digestion, by electrophoresis, cuts glue
It after recycling, is connected overnight with T4 DNA ligase, connection product transformed competence colibacillus cell E.coli DH5a, coating Amp plate is simultaneously
The identification of picking single colonie, identifies that the plasmid of correct positive colony is named as pcDNA5/FRT-IL6.
4. for improving the additive of hybridoma cell clone quantity, which is characterized in that any described using claim 1-3
Preparation method preparation.
5. the culture medium of hybridoma, which is characterized in that be in the DMEM containing 10% fetal calf serum added with claim
Additive described in 4, the volume ratio of the additive of addition are 20%.
6. the method for cultivating hybridoma, which is characterized in that add the claim that volume ratio is 20% in its culture medium
Additive described in 4, or using culture medium described in claim 5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610005108.9A CN105462934B (en) | 2016-01-05 | 2016-01-05 | For improving the additive and preparation method thereof of hybridoma cell clone quantity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610005108.9A CN105462934B (en) | 2016-01-05 | 2016-01-05 | For improving the additive and preparation method thereof of hybridoma cell clone quantity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105462934A CN105462934A (en) | 2016-04-06 |
| CN105462934B true CN105462934B (en) | 2019-04-05 |
Family
ID=55601095
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610005108.9A Active CN105462934B (en) | 2016-01-05 | 2016-01-05 | For improving the additive and preparation method thereof of hybridoma cell clone quantity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105462934B (en) |
Families Citing this family (3)
| 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 |
| 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 |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898041A (en) * | 2012-12-25 | 2014-07-02 | 深圳先进技术研究院 | Culture method of hybridomas |
-
2016
- 2016-01-05 CN CN201610005108.9A patent/CN105462934B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103898041A (en) * | 2012-12-25 | 2014-07-02 | 深圳先进技术研究院 | Culture method of hybridomas |
Non-Patent Citations (1)
| Title |
|---|
| 人白介素6在CHO细胞中的克隆与表达;韩素文等;《军事医学科学院院刊》;19970228;第21卷(第1期);第32页左栏第19-20行，右栏倒数第3段 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105462934A (en) | 2016-04-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103172749B (en) | Preparation of African swine fever protein engineering vaccine | |
| CN105462934B (en) | For improving the additive and preparation method thereof of hybridoma cell clone quantity | |
| CN100391969C (en) | Helicobacter Pylori urease B subunit Th epitope peptide, its coding DNA, vaccine and uses | |
| CN109971726B (en) | Hybridoma cell strain, antibody produced by hybridoma cell strain and preparation method of antibody | |
| CN103193887A (en) | Recombinant porcine IL2-Fc (interteukin-2-Fc) fusion protein as well as encoding gene and expressing method of fusion protein | |
| CN108558997A (en) | A kind of application of recombination fusion protein TIGIT-Fc and its resisting transplant rejection reaction | |
| CN104965083B (en) | A kind of kit of detection H3N2 hypotype canine influenza virus | |
| CN108250293B (en) | anti-Ebola virus VP40 protein monoclonal antibody G7A6 and application thereof | |
| CN106086067A (en) | The recombinant plasmid of expression J subgroup avian leucosis virus gp85, p27, p10 gene of ubiquitin mediation and construction method thereof and application | |
| CN101671655A (en) | Monoclonal antibody hybridoma cell of HIV P24 and application | |
| CN105132376B (en) | One can the how individual epitopes of specific recognition HBsAg monoclonal antibody and its application | |
| CN103509815A (en) | Preparation method of recombinant panda IL-2 immune adjuvant | |
| CN103820459B (en) | A kind of algae feed and application of energy birds flu-preventing | |
| CN104059941A (en) | Fusion gene eukaryotic expression construct, and genetic engineering vaccine and preparation method thereof | |
| CN106047810A (en) | Novel DC-CTLs cell culture system and culture method thereof | |
| CN103409453A (en) | Preparation method of recombinant panda IL-6 immunological adjuvant | |
| CN101438977A (en) | Method for constructing HSAT H-T-S mouse-human chimeric model and application | |
| CN110227153A (en) | A kind of preparation method and applications of porcine rotavirus subunit vaccine | |
| CN108611371A (en) | The preparation method of the Goats Milk gland bioreactor of GP5-M albumen is expressed in milk | |
| CN107880135A (en) | One breeder luteinizing hormone releasing hormone recombinant antigen castration vaccine and preparation method thereof | |
| CN104292310B (en) | Duck plague virus UL15 gene exonI recombinant proteins and its preparation method and application | |
| CN102604979A (en) | Nucleotide sequence, fusion protein coded by nucleotide sequence, Mycobacterium tuberculosis vaccine prepared based on nucleotide sequence and preparation method of Mycobacterium tuberculosis vaccine | |
| CN103243119B (en) | The expression of Soluble CD83 and preparation method | |
| CN110713540B (en) | Hybridoma cell strain secreting monoclonal antibody against large yellow croaker immunoglobulin T | |
| CN1173741C (en) | Concatenated antidiotype monoclonic antibody vaccine for fish diseases |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |