CN103305469B - Serum-free culture adapted recombinant protein high-expression cell strain QB-Tn9-4S-Cl-F and application thereof - Google Patents
Serum-free culture adapted recombinant protein high-expression cell strain QB-Tn9-4S-Cl-F and application thereof Download PDFInfo
- Publication number
- CN103305469B CN103305469B CN201310204918.3A CN201310204918A CN103305469B CN 103305469 B CN103305469 B CN 103305469B CN 201310204918 A CN201310204918 A CN 201310204918A CN 103305469 B CN103305469 B CN 103305469B
- Authority
- CN
- China
- Prior art keywords
- serum
- cell
- virus
- recombinant protein
- free
- 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.)
- Expired - Fee Related
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a serum-free culture adapted recombinant protein high-expression cell strain QB-Tn9-4S-Cl-F and an application thereof. The microbe preservation number of the cell strain disclosed by the invention is CCTCC (China Center for Type culture Collection) NO:C201327. The serum-free culture adapted cell strain obtained by the invention has no obvious difference in the aspects of morphology, growth characteristics and sensitivity to virus after being serially passed for 50 generations in an Sf900III S FM (abbreviated to Sf900 III) culture medium compared with that in a culture medium containing 10 percent serum, but the expression level of the serum-free cultured clone strain recombinant protein is remarkably improved compared with that in the complete medium containing 10 percent serum, so that the problems of purification and post treatment of genetic engineering expression products are avoided, and broad prospect in exogenous gene active protein industrial production is present; meanwhile, the cell clone strain disclosed by the invention has high sensitivity to virus, and provides a good approach for large-scale low-cost production of viral insecticide as the yield of the cells strain is similar to that in a serum medium.
Description
Technical field
The present invention relates to the insect cell line of serum-free culture, particularly relate to cabbage looper cell clone QB-TN9-4S-CL-F(be called for short QB-Tn9-CL-F) serum-free culture.Under the invention still further relates to serum-free condition, this cell clone is preparing the purposes in viral pesticide and recombinant protein production, belongs to cytobiology and biological technical field.
Background technology
Since first insect cell line is in the world set up (Grace1962), Insect cellculture has been widely used in Basic of Biology research and production field.Utilize baculovirus to the infection of insect cell, massive duplication virus is as biotic pesticide, and the product that its product and living worm body are produced compares and has lot of advantages (Wise and uaughn, 1986; Granados efal.1987).Particularly baculovirus--the foundation of insect cell expression vector system, by virus infection insect cell, the foreign gene that great expression virus is carried, Restruction albumen (Luckow and Summers, 1988).Due to this system have efficiently, safety, capacity are large, the product of expressing has the advantages such as biological activity, so the cell cultures of insect is all the noticeable carrier (Ping Wang.efal.1992) having very much development potentiality in the production of viral pesticide or the exogenous genes products expression in biology, medical science and agriculture field.
Current people have utilized several insect cell line successful expression and production various insects viral pesticide, vaccine and medical product (Granados1994; Altman etal.1999) (Granados efal.2007).Usual Insect culture medium contains the foetal calf serum (FBS) of 10%, to provide the necessary nutrition of Growth of Cells.Because foetal calf serum is expensive and limit the in vitro a large amount of of viral pesticide and produce; In Restruction protein process, the existence of serum in substratum, also makes the product purification of expression and reclaims to cause very large difficulty, on the other hand, in virus replication phase, the substratum of low serum or serum-free contributes to the expression level or the polyhedrin production that improve recombinant protein.The stable cell strain adapting to serum-free culture of exploitation just becomes the key problem in technology reducing costs and improve output.For this reason, the serum-free culture developing serum free medium and cell is the study hotspot of Insect cellculture engineering field always.But the report of this respect is very few, report Sf9(Spodoptera frug in the eighties; Perda) growth characteristics (the Maiorala et.al.1988 of cell strain serum-free culture; Inlow et.al.1989), have the clone of report BTI-Tn5B1-4 and HzAm1(bollworm recently) serum-free of cell or the domestication of low serum (Granados et.al.1995 Dai Hu etc., 2000; Zhang Youhong etc., 2005).
In these researchs, there is no virus after being reported in serum-free culture insect cell Long Term Passages output and exogenous genes products are expressed.The present inventor has successfully tamed the new cell clone QB-TN9-4S-CL-F of a strain.This clone strain go down to posterity for a long time in serum-free culture (P.50) showed the superperformance of stable high yield high expression level afterwards.
Summary of the invention
The technical problem to be solved in the present invention tames out a strain to keep vigorous vitality under serum-free culturing conditions, has viral high yield, the cell of recombinant protein high expression level.
Technical problem to be solved by this invention is achieved through the following technical solutions:
The cabbage looper cell clone of a strain serum-free culture of the present invention, called after QB-TN9-4S-CL-F, be called for short QB-Tn9-CL-F, its microbial preservation number is: CCTCC NO:C201327; Classification And Nomenclature is: Trichopllusia ni cell line; Shelf time is on April 9th, 2013; Preservation unit is: China typical culture collection center; Preservation address is: Wuhan University.
It is high that the stable cell adapting to serum-free culture can overcome the production cost that expensive serum causes, the difficult problem in the cell cultures engineering fields such as expression product is purifying not easily, and expression level is low.The present invention utilizes multiple serum free medium (TC-100 for this reason, Grace ' s, Sf900 III) test screen in different cell strains, repeat to cultivate, screen through the serum that repeatedly successively decreases, final acquisition can be called for short Sf900III at Sf900 III SFM() the medium-term and long-term stable cultured cells strain of substratum.Cell strain continuous passage more than the 50 times well-growns in Sf900 III substratum of the adaptation serum-free culture that the present invention obtains, feature is stablized, with containing when cultivating in 10% blood serum medium without significant difference.On expression level, the QB-Tn9-CL-F clone strain expression of recombinant proteins level of serum-free culture significantly improves than when cultivating in perfect medium, owing to it also avoid the purifying of gene engineering expression product and a difficult problem for aftertreatment without foetal calf serum.Therefore in foreign gene activity albumen suitability for industrialized production, wide prospect is illustrated.
In addition, the cabbage looper cell clone that the invention allows for described adaptation serum-free culture is preparing the purposes in desinsection virus or recombinant protein.
When for the preparation of desinsection virus, directly can utilize the cell clone described in desinsection virus infection, carry out serum free suspension cultivation, carry out a large amount of productions of desinsection virus.
Described desinsection virus comprises can infect all insect viruses of this cell, and preferably, the desinsection of the viral clover californica nuclear polyhedrosis virus (AcMNPV) or other kind for can be used as biotic pesticide of described desinsection is viral.
The cell clone of serum-free culture of the present invention is extremely sensitive to AcMNPV, and output can similar at serum free culture system, produces viral pesticide provide good approach for large-scale low-cost.
When for the preparation of recombinant protein, comprise the expression vector or recombinant virus that build containing goal gene, this recombinant expression vector be directly transformed into described cell clone or infect described cell clone with recombinant virus, then carrying out serum free suspension cultivation, Restruction albumen.
Described recombinant protein comprises beta-galactosidase enzymes (β-galactoridase) or recombinant basic Phosphoric acid esterase (SEAP); Or agricultural, medicine and the albumen required for other field.
Cell strain of the present invention is cultivated and is compared with in containing the perfect medium of 10% serum in serum free medium, recombinant protein SEAP and nearly 2 times of β-gal output increased, so cell strain of the present invention is also the good carrier that other recombinant proteins are produced.
Accompanying drawing explanation
Fig. 1 is the form of QB-Tn9-CL-F cell;
A: cultivate (the 86th generation) in perfect medium (TNM-FH); B: cultivated for 20 generations in serum free medium (Sf900 III); C: cultivated for 30 generations in serum free medium; D: cultivated for 50 generations in serum free medium;
Illustrate QB-Tn9-CL-F clone go down to posterity for a long time in serum free medium after its form with cultivate without significant difference in the perfect medium (TNM-FH);
Fig. 2 is that the growth curve of Tn-5B1-4 and the Sf-9 cell that the QB-Tn9-CL-F cell of serum-free culture is cultivated with perfect medium compares:
Illustrate that in the growth velocity of the QB-Tn9-CL-F cell of serum-free culture and perfect medium, Tn-5B1-4 cell (commodity are called HighFive, the excellent cell of current widespread use) is similar, and be significantly higher than the Sf-9 cell that perfect medium cultivates;
Fig. 3 is the QB-Tn9-CL-F cell infecting AcMNPV virus;
A, C are respectively 20 generations and the cell infection of 50 generation cells in perfect medium;
B, D are respectively 20 generations and the cell infection of 50 generation cells in serum free medium;
Fig. 4 is that the β-gal. of each clone when growing in serum-free and perfect medium respectively expresses:
Illustrating that the QB-Tn9-CL-F cell β-gal. of serum-free culture expresses the cell be significantly higher than in other perfect mediums, is in perfect medium 1.8 times of this cell in the infection expression of the 8th day;
Fig. 5 is that the SEAP of each clone when growing in serum-free and perfect medium respectively expresses:
Illustrate that the QB-Tn9-CL-F cell SEAP of serum-free culture expresses just to be significantly higher than all cells in other perfect mediums at the 7th day and to express, its expression amount is about in perfect medium 2.0 times of this cell.
Embodiment
The present invention is further described below in conjunction with specific embodiment.Advantage and disadvantage of the present invention will be more clear along with description.But these examples are implemented just exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
The adaptive process of embodiment 1 serum-free culture insect cell QB-Tn9-CL-F
The adaptive process of 1.1 serum-free culture:
First will from the clone strain of the Trichopllusia ni cell line QB-TN9-4S of perfect medium (containing 10%FBS) logarithmic phase (by 2.0 × 10
5cells/ml concentration) go down to posterity in several serum free medium (TC-100 containing different concns serum, EX CELL-420, SF-900III) cultivate in, adjusting serum-concentration in substratum during beginning is 5%, 28 DEG C of quiescent culture, every day observation of cell upgrowth situation, until Growth of Cells to after at the bottom of being paved with bottle, cell is blown and beaten into suspension, measure cell survivaling number, go down to posterity by above-mentioned cell concn again according to viable cell ratio, when cytoactive is stabilized in more than 90%, then cell is imported in the substratum containing 2.5%FBS.Use the same method make cell 2.5%, 1.25%, adapted in the substratum of 0.625%FBS, finally obtain and grow stable cell strain in the substratum SF-900 III of serum-free.This is the first-generation cell strain of serum-free culture.
After 1.2 Long Term Passages, cell stability measures:
Propagating method routinely, by the 5ml first-generation 2.0 × 10
5the cell of cells/ml serum-free culture accesses 25 ㎝
2in culturing bottle, 28 DEG C of cultivations, measured viable cell concentrations after three days.Repetition like this 50 times, after going down to posterity at every turn observation of cell upgrowth situation and measure viable counts record every five times.Measured for the 50th generation, result can be found out, after long-term cultivation, the cell bio-activity of this serum-free culture is more stable.
The strain that the present invention obtains adapts to the cabbage looper cell clone of serum-free culture, called after QB-TN9-4S-CL-F, and be called for short QB-Tn9-CL-F, its microbial preservation number is: CCTCC NO:C201327; Shelf time is on April 9th, 2013; Preservation unit is: China typical culture collection center; Preservation address is: Wuhan University.
1.3. cell clone form and biological characteristics
A, cellular form: cell, in Sf900 III serum free medium, is cultivated 3 days under 28 DEG C of conditions, on the occasion of logarithmic phase, under inverted phase contrast microscope (Olympus, 1 × 71Papam), observation of cell form, measures size.
Result: cellular form, size and in perfect medium without significant difference.Spindle cell accounts for more than 80%, and size is 18.4 × 49.4 μm.
The QB-Tn9-CL-F cellular form in the 20th generation of cultivating in the serum free medium (Sf900 III), 30 generations and 50 generations is contrasted with the form of the 86th generation QB-Tn9-CL-F cell cultivated in perfect medium (TNM-FH), result show QB-Tn9-CL-F clone go down to posterity for a long time in serum free medium after its form with cultivate without significant difference in the perfect medium (TNM-FH), result is as shown in Figure 1.
B. Growth of Cells: by method (1994, the J.Invert. of Robert R.Granados.
Pathol., 64:260-266), measure cell growth curve and population doubling time.By the cell (P.50) of logarithmic phase by 1.0 × 10
6the concentration access 25cm of cell/5ml substratum
2in culturing bottle, cultivate under 28 ° of C conditions, every 24 hours sampling and measuring cell concns.Draw cell growth curve according to record result, and press formula T=1/K (log N=Log N
0+ K+log2) calculate population doubling time (T).
Result: as can be seen from the growth characteristics of cell, this cell clone well-grown in serum free medium, compares without significant difference with the Tn5B1-4 in perfect medium, and Growth of Cells maximum density reached 1.90 × 10 respectively at the 5th day
6cell/ml and 2.0 × 10
6cell/ml, its population doubling time is respectively 21.9hr and 21.5hr.In serum free medium Sf900 III and in the perfect medium (TNM-FH) containing 10% foetal calf serum, the growth velocity of QB-TN9-CL-F cell strain and Sf-9 cell (TNM-FH substratum) compares obvious advantage (population doubling time of Sf-9 cell is 25.4hr), and result as shown in Figure 2.
Virus infection titer (the TCID of embodiment 2 clone strain of the present invention
50) and polyhedron (OB) determination of yield
According to the method (1992 of Ping Wang, J.Invert.Pathol., 59:46-53), utilize AcMNPV-1A (Autographa californica multicapsid nucleopolyhedrosisvirus nuclear polyhedrosis virus) for malicious source (Boyce Thomason Institate, Cornell University, Ithaca, NY, USA), with MOI=10(Multiplicity of Infection) infestation index's inoculation logarithmic phase cell, cultivate under 28 ° of C, every day observes infection state, suspended after 3 days, after centrifugal, Supernatant samples is for measuring titre; Cultivate after 4 days, polyhedron is fully formed, and when cell not yet breaks, examines under a microscope and calculates infection rate.Finally use ultrasonic treatment cell, after discharging whole polyhedron, polyhedron output (OBs/ml) can be obtained with blood cell computing board counting.
Test-results: cell clone QB-TN9-4S-CL-F(of the present invention is referred to as QB-Tn9-CL-F) in serum free medium to AcMNPV viral susceptibility with compare no significant difference in perfect medium.Within 4 days, measure after infection.Infection rate is on average more than 95%.Now existing 30% lysis in serum free medium, and discharge polyhedron in substratum.In two kinds of substratum, venereal disease poison (BV) volume variance that sprouts of cell is not remarkable, is about 5.0 × 10
7tCID
50/ ml.The polyhedron of cell in serum free medium (OB) output is a little more than the cell in perfect medium, on average be respectively 93.9OBs/ cell and 91.4OBs/ cell, illustrate that the present invention provides good approach, Fig. 3 and table 1 for reducing viral pesticide production cost.
Infection rate in table 1 different culture media and viral yield
The expression test of embodiment 3 recombinant protein in the cell clone QB-Tn9-4s-CL-F of serum-free culture of the present invention
One, test method
Clone:
Sf-9 clone, QB-Tn9-CL-A clone, QB-Tn9-CL-B clone, QB-Tn9-CL-C clone, QB-Tn9-CL-F clone, High Five(BTI-Tn5B1-4) clone.
According to method (1992, Bio/technology, the 10:1148-1150 of Wickham and David; 1993, In Nitro Cell Dev.Biol.29A:388-390), recombinant protein alkaline phosphatase (SEAP) and tilactase (β-gal.) expression level determined.
1, the mensuration of β-gal. expression:
The cell of logarithmic phase is divided into two groups, by 2.0 × 10
5the amount in cell/2ml/ hole accesses in 24 orifice plates, uses the substratum of serum-free (Sf900 III) and perfect medium (TNM-FH) to cultivate respectively, until at the bottom of cell precipitation hole time, absorbs substratum supernatant gently.By the Index of infection access recombinant virus AcMNPV-β-gal of MOI=10, every 24 h apart samplings, at OD
420optical density(OD) under measure density value, expression level (IU/ml) can be calculated according to following formula.
2, the mensuration of SEAP expression:
Sample is prepared according to above-mentioned steps (β-gal. assay method), then with same Index of infection access recombinant virus AcMNPV-SEAP for examination cell.Every 24 h apart samplings.Under OD405 optical density(OD), measure density value, measure expression level (IU/ml) according to following formula.
Two, test-results
1, the expression level of beta-galactosidase enzymes (β-gal.)
Cell clone QB-Tn9-CL-F of the present invention cell in serum-free culture compares with cell in perfect medium, shows significant high expression level.In serum-free culture, the β-gal of cell was about 1.8 times (Fig. 4) of cell in perfect medium at the 8th day.
2, the expression level of alkaline phosphatase (SEAP)
Cell clone QB-Tn9-CL-F of the present invention cell in serum-free culture produced the output of SEAP apparently higher than other cells grown in perfect medium at the 7th day, can reach about 2.0 times (Fig. 5).
From above-mentioned two kinds of expression of recombinant proteins levels, also prove that low serum is conducive to the theory of the expression of exogenous genes products and the production of polyhedrin.
Claims (7)
1. a strain adapts to the cabbage looper cell clone of serum-free culture, called after QB-TN9-4S-CL-F, and be called for short QB-Tn9-CL-F, its microbial preservation number is: CCTCC NO:C201327; Shelf time is on April 9th, 2013; Preservation unit is: China typical culture collection center; Preservation address is: Wuhan University.
2. the cabbage looper cell clone of adaptation serum-free culture according to claim 1 is preparing the purposes in desinsection virus or recombinant protein.
3. according to purposes according to claim 2, it is characterized in that: directly utilize the cell clone described in desinsection virus infection, utilize serum free medium Sf900 III SFM suspension culture, carry out a large amount of productions of desinsection virus.
4. according to purposes according to claim 2, it is characterized in that: build the expression vector containing goal gene or recombinant virus, this recombinant expression vector is directly transformed in described cell clone or with recombinant virus and infects described cell clone, then serum free medium Sf900 III SFM is utilized to carry out suspension culture, Restruction albumen.
5. according to purposes according to claim 4, it is characterized in that: described recombinant protein is beta-galactosidase enzymes.
6. according to purposes according to claim 4, it is characterized in that: described recombinant protein is alkaline phosphatase.
7. according to purposes according to claim 3, it is characterized in that: described desinsection virus is the desinsection virus of clover californica nuclear polyhedrosis virus or other kind.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310204918.3A CN103305469B (en) | 2013-05-28 | 2013-05-28 | Serum-free culture adapted recombinant protein high-expression cell strain QB-Tn9-4S-Cl-F and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310204918.3A CN103305469B (en) | 2013-05-28 | 2013-05-28 | Serum-free culture adapted recombinant protein high-expression cell strain QB-Tn9-4S-Cl-F and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103305469A CN103305469A (en) | 2013-09-18 |
| CN103305469B true CN103305469B (en) | 2015-01-07 |
Family
ID=49131191
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310204918.3A Expired - Fee Related CN103305469B (en) | 2013-05-28 | 2013-05-28 | Serum-free culture adapted recombinant protein high-expression cell strain QB-Tn9-4S-Cl-F and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103305469B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108531442B (en) * | 2018-04-28 | 2021-10-01 | 青岛农业大学 | Insect cell line cultured in serum-free suspension manner and application thereof |
| CN112980767B (en) * | 2021-02-08 | 2023-09-26 | 东莞博盛生物科技有限公司 | Norda virus-free monoclonal insect cell line and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6403375B1 (en) * | 1994-08-24 | 2002-06-11 | Boyce Thompson Institute For Plant Research, Inc. | Establishment of Trichoplusia ni cell lines in serum-free medium for recombinant protein and baculovirus production |
| US6342216B1 (en) * | 1999-03-17 | 2002-01-29 | The Board Of Regents, The University Of Texas System | Therapy of cancer by insect cells containing recombinant baculovirus encoding genes |
-
2013
- 2013-05-28 CN CN201310204918.3A patent/CN103305469B/en not_active Expired - Fee Related
Non-Patent Citations (2)
| Title |
|---|
| Establishment of New Trichoplusia ni Cell Lines in Serum-Free Medium for Baculovirus and Recombinant Protein Production;Kevin A. McKenna et al.;《JOURNAL OF INVERTEBRATE PATHOLOGY》;19981231;全文 * |
| Studies on Serum-Free Culture of Insect Cells for Virus Propagation and Recombinant Protein Production;PINGWANG et al.;《JOURNALOFINVERTEBRATEPATHOLOGY》;19921231;全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103305469A (en) | 2013-09-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103667176B (en) | Carassius auratus gibelio brain tissue cell line sensitive to cyprinid herpesvirus II, and establishing method and application thereof | |
| CN102604889B (en) | HEK (human embryonic kidney) 293 cell line applicable to serum-free culture and application thereof | |
| CN103275925B (en) | Construction method of mandarin fish brain cell system | |
| WO2020259532A1 (en) | Method for producing virus and harvest liquid composition | |
| CN103305469B (en) | Serum-free culture adapted recombinant protein high-expression cell strain QB-Tn9-4S-Cl-F and application thereof | |
| CN107523555A (en) | The method for obtaining virus | |
| CN104974977A (en) | Epinephelus lanceolatus kidney tissue cell line and construction method thereof | |
| CN108531442B (en) | Insect cell line cultured in serum-free suspension manner and application thereof | |
| CN104087549B (en) | The insect cell line of high yield baculovirus and application thereof | |
| CN101935634B (en) | Clonal strain of cabbage looper cell line and application thereof | |
| CN101070533A (en) | American cotton bollworm yong-insect fatbody cell line of high yield stab-like virus, its constitution and use | |
| CN109402068A (en) | A method of preparing the remaining porcine pseudorabies virus of serum-free | |
| CN105950571B (en) | A kind of enrichment procedure of the mandarin fish infectious spleen and kidney necrosis virus ISKNV based on carp epithelium oncocyte EPC | |
| CN112980767B (en) | Norda virus-free monoclonal insect cell line and application thereof | |
| CN108823205A (en) | A kind of HEK293T cell line construction method knocking out PLAC8 gene | |
| CN106047932B (en) | Methyl-B-cyclodextrin is improving the application in baculoviral exogenous protein expression amount | |
| CN106939318A (en) | A kind of single cell clone separation method | |
| CN110387357A (en) | A kind of purification process of the recombinant virus with fluorescent marker | |
| CN104774873A (en) | Grass carp reovirus in-vitro assembly preparation method | |
| CN102453698A (en) | Method for suspension culture of subculture cells and method for producing hog cholera vaccine by using subculture cells | |
| CN112359064B (en) | Application of methyl-beta-cyclodextrin in improving transduction efficiency of baculovirus to mammalian cells | |
| CN103555675B (en) | Insect cell High Five application in cultivating Antheraea pernyi nuclear polyhedrosis virus | |
| CN112760277A (en) | Oriental myxozoon pupa ovarian cell line for high yield of baculovirus and preparation method and application thereof | |
| CN104805053A (en) | Pearl giant grouper proboscis tissue cell line as well as constructing method and application thereof | |
| US5405770A (en) | Heliothis subflexa cell line for the production of baculoviruses |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20150107 Termination date: 20160528 |