CN106367379A - Insect cell maintenance liquid for increasing expression amount of hog cholera virus E2 recombinant proteins and preparation method thereof - Google Patents
Insect cell maintenance liquid for increasing expression amount of hog cholera virus E2 recombinant proteins and preparation method thereof Download PDFInfo
- Publication number
- CN106367379A CN106367379A CN201611113323.7A CN201611113323A CN106367379A CN 106367379 A CN106367379 A CN 106367379A CN 201611113323 A CN201611113323 A CN 201611113323A CN 106367379 A CN106367379 A CN 106367379A
- Authority
- CN
- China
- Prior art keywords
- liquid
- insect cell
- cell
- phosphate
- cell maintaining
- 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.)
- Granted
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
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0601—Invertebrate cells or tissues, e.g. insect cells; Culture media therefor
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/50—Soluble polymers, e.g. polyethyleneglycol [PEG]
-
- 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
- C12N2501/90—Polysaccharides
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to the technical field of biology, particularly relates to an insect cell maintenance liquid for increasing the expression amount of hog cholera virus E2 recombinant proteins and a preparation method thereof. To be specific, baculovirus is specifically added in an equilibrium liquid-phosphate buffer solution(1 * PBS) to synthesize the limiting amino acids of E2 proteins, dextran sulfate sodium and polyether F-68 which are used as the maintenance liquid to replace an insect cell cell culture medium used for inoculating recombinant baculovirus. With the same culture conditions, same cell quantity and same virus-inoculating quantity, compared with the insect cell culture medium, the insect cell maintenance liquid for increasing the expression amount of hog cholera virus E2 recombinant proteins increases the infection of cells within an unit quantity, the production quantity of the recombinant baculovirus and the secretion amount of the E2 recombinant proteins.
Description
Technical field
The invention belongs to biological technical field, it is specifically related to a kind of elder brother improving swine fever virus e2 expression of recombinant proteins amount
Worm cell maintenance medium and its compound method.
Background technology
Baculovirus expression system correctly can be folded in the cell for the foreign protein of high expression, the collocation of disulfide bond
And the formation of oligomer provides good environment, expression product can be made in structure and functionally close to native protein, so utilizing
Insect cell-baculovirus expression system recombiant protein is one of important directions of subunit vaccine research and development.But, due to insecticide
Cell culture medium is expensive, and expression of recombinant proteins amount is relatively low, leads to a lot of recombinant baculovirus subunit vaccines to be stopped in reality
Test room development.Conventional insect cell-baculovirus expression system connects malicious method is, when insect cell growth is to necessarily close
Degree, when motility rate is not less than 95%, adjusts cell density with insect cell medium, inoculates baculoviruss after supplementing system nutrient.
The serum-free insect cell culture medium of commercialization is expensive, and, between 6.10-6.40, osmotic pressure is between 350-380mosm/ for ph value
kg.The method does not consider to connect the restrictive factor of cell growth demand and virus replication after poison, not only impact swine fever virus e2 weight
The expression of histone, and unit volume antigen production cost is high.
Current study show that, its reason is probably, one, the propagation with insect cell, energy, limiting amino acid are needed
The amount of asking increases, and cell metabolite accumulates (lactic acid, alanine, uric acid, nh in a large number simultaneously4+Deng), the ph of culture fluid, osmotic pressure are corresponding
Change, greatly impact insect cell growth and the propagation connecing malicious restrovirus.When cell culture system lacks L-Glutamine
Cell nucleic acid, protein can be affected, so that impact cell growth.But unstable L-Glutamine can be to culture in solution
Cell produces adverse effect, and Alanyl-Glutamine (glutamine dipeptide, ala-gln) is a kind of dipeptides, and good stability, in culture medium
Middle can effectively replace L-Glutamine, promotes cell growth.Culture medium during the report Insect cellculture such as kennie u.dee
The glucose providing disclosure satisfy that demand, and aspartic acid consumption is more, is one of restraining factors of cell growth.2nd, recombinate
Baculovirus infection insect cell Early manifestation increases for cell dia, and nucleus become big；Late manifestations stop growing for cell,
Virus is sprouted, partial-length apoptosis, infection end-stage cells cracking.Later period of infection is the optimum harvest date of recombiant protein.Virus profit
During host cell synthesis oneself protein, may be because of the propagation of the disappearance impact virus of the nutrients such as system aminoacid, duplication.
Content of the invention
It is an object of the invention to: provide a kind of insect cell improving swine fever virus e2 expression of recombinant proteins amount and maintain
Liquid and its compound method, for regulation system component after insect cell inoculation recombinant baculovirus, supplement nutrient, to maintain cell
Growth, promotes the propagation of recombinant baculovirus and the secretory volume of swine fever virus e2 recombiant protein, reduces expression cost simultaneously.
The present invention technical scheme: a kind of insect cell maintaining liquid of raising swine fever virus e2 expression of recombinant proteins amount,
Described insect cell maintaining liquid is to be gathered by interpolation limiting amino acid, sulphuric acid Portugal in 1 × pbs balance liquid-phosphate buffer
Sugared sodium salt and polyethers f-68 composition.
Further, described 1 × pbs balance liquid-phosphate buffer is by sodium chloride 6000-10000mg/l, potassium chloride
100-300mg/l, the concentrated hydrochloric acid composition of disodium hydrogen phosphate 1000-2000mg/l, potassium dihydrogen phosphate 100-400mg/l and surplus,
The concentrated hydrochloric acid of described surplus refers to the ph value of above-mentioned 1 × pbs balance liquid-phosphate buffer is adjusted to 6.40 usage amount.
Further, described 1 × pbs balance liquid-phosphate buffer is by sodium chloride 8000mg/l, potassium chloride 200mg/l, phosphoric acid hydrogen
The concentrated hydrochloric acid composition of disodium 1440mg/l, potassium dihydrogen phosphate 240mg/l and surplus, the concentrated hydrochloric acid of described surplus refers to above-mentioned 1
The ph value of × pbs balance liquid-phosphate buffer is adjusted to 6.40 usage amount.
Further, described limiting amino acid refers in glycine, dl- threonine, l- L-Valine, l- leucine, l-
Aspartic acid, l- agedoite and glutamine dipeptide.Further, described glycine 20-40mg/l, dl- threonine 80-
105mg/l, l- L-Valine 80-105mg/l, l- leucine 90-150mg/l, l- aspartic acid 20-40mg/l, l- agedoite
30-70mg/l, glutamine dipeptide 150-450mg/l.Further, described glycine 30mg/l, dl- threonine 95mg/l, l-
L-Valine 94mg/l, l- leucine 105mg/l, l- aspartic acid 30mg/l, l- agedoite 50mg/l and glutamine dipeptide
300mg/l.
Further, described sodium dextran sulfate 80-120mg/l；Described polyethers f-68 is 800-1200mg/l.More enter one
Step, described sodium dextran sulfate 100mg/l；Described polyethers f-68 is 1000mg/l.
The method preparing above-mentioned insect cell maintaining liquid: deionized water prepares 1 × pbs balance liquid-phosphate buffer,
Deca concentrated hydrochloric acid adjusts ph to after 6.40；Add glycine, dl- threonine, l- in above-mentioned balance liquid-phosphate buffer
L-Valine, l- leucine, l- aspartic acid, l- agedoite, glutamine dipeptide, sodium dextran sulfate and polyethers f-68, fully molten
Filtered with 0.22 μm of sterilizing filter after solution；Finally, steriling test is qualified, and osmotic pressure in the solution of 350-380mosm/kg is
Can be used as insect cell maintaining liquid.
Beneficial effect: the present invention, according to genetic algorithm, analyzes 373 aminoacid constituting swine fever virus e2 albumen, its
Middle amino acid number most for L-Valine, threonine, leucine, glycine, be recombinant baculovirus synthesis e2 recombiant protein
Limiting amino acid.Wherein, dextran sulfate sodium salt can effectively facilitate insect cell suspension culture and the expression of albumen；Polyethers
F-68 can protect the infringement that the cell in suspension causes from transfer and stirring, carries out high-volume cell culture in fermentation tank
When, can prevent air from adhering to cell, stablize cell surface foam to improve the resistance that cell membrane is sheared to hydrodynamic force.
Present invention specificity in balance liquid-phosphate buffer (1 × pbs) adds the limit that baculoviruss synthesize e2 albumen
Acidic amino acid processed, dextran sulfate sodium salt and polyethers f-68, are used for substituting the insect cell inoculation shaft-like disease of restructuring as maintaining liquid
The cell culture medium of poison.Identical condition of culture, cell quantity and connect poison amount under, compared with insect cell medium, this
Bright significantly improve Board Lot cell infection, produce recombinant baculovirus amount and secretion e2 recombiant protein content, largely
On reduce production cost.
Specific embodiment
With reference to instantiation, the invention will be further described, but not as a limitation of the invention.
Embodiment 1, it is binned in Csfv E 2 sequence on baculoviruss with the analysis of biological software dnaman
(1119bp) and its coding aminoacid sequence (373aa), compare the amino acid composition of sequence, predict the isoelectric point, IP of this albumen
For 5.93, the results are shown in Table 1.Based on genetic algorithm predict this albumen synthesis major limitation acidic amino acid be L-Valine, threonine,
Leucine, glycine.
Table 1 swine fever virus e2 histone amino acid constituents
Insect cell growth metabolism result of study shows, aspartic acid quickly consumes with cell growth；Based on laboratory to elder brother
Worm cell suspension cultures research, it was demonstrated that interpolation polyethers f-68 and dextran sulfate sodium salt can improve cell anti-shearing force, improves thin
Born of the same parents' suspension culture state.
To sum up, the cell maintenance medium after the application establishes a kind of inoculation recombinant baculovirus for insect cell is prepared new
Method, adds cell culture protective agent polyethers f-68, dextran sulfate sodium salt in 1 × pbs balance liquid and can improve weight
The specific amino acid of histone expression, concrete compound method is as follows:
Following reagent is weighed according to table 2, deionized water prepares 1 × pbs balance liquid, Deca concentrated hydrochloric acid adjusts ph to after 6.40
Constant volume, standby.
Table 2 pbs balances formula of liquid
Following reagent is weighed according to table 3, is dissolved in successively in freshly prepared 1 × pbs balance liquid (ph=6.40), with 0.22 μm
Sterilizing filter filter, keep sample and carry out steriling test and osmometry.Steriling test is qualified, and osmotic pressure is between 350-
The solution of 380mosm/kg can be used as the maintaining liquid after insect cell inoculates baculoviruss, for improving cell suspension cultures bar
Part, improves the expression of swine fever virus e2 recombiant protein.
Table 3 insect cell connects poison and maintains formula of liquid
When insect cell growth to certain density, add volume required viral maintaining liquid, adjustment cell density inoculates shaft-like disease
Poison, in virus infection peak period (late period), sampling carries out cell counting；After harvesting virus, swine fever virus e2 weight in detection venom
The quantitation of histone.
Practical proof, 1, sf9 cell suspension cultures expression e2 albumen sf-9 cell line come from United States Department of Agriculture's insecticide pathology
Laboratory, from the ovary tissue of autumn fly anthelmintic (Spodopterafrugiperda spodoptera frugiperda) pupa.high five
Cell (bti-tn-5b1-4) comes from the clone and separate strain of cabbage looper (trichopulsia ni) parental cell line.By
It is very sensitive to model virus autographa california nuclear polyhedrosis virus (acmnpv) in sf-9 and high five cell strain,
Thus become the commonly used cell line for baculovirus expression of each laboratory.
Sf-9 cell is carried out suspension culture with sf900ii serum-free insect cell culture fluid, when cell density reaches 2.0
×106During individual/ml (cell survival rate is not less than 95%), by cell equivalent sub-bottle cultivate, respectively use insect cell medium and
Viral maintaining liquid adjustment cell density is to 1.0 × 106Individual/ml, inoculates equivalent swine fever virus e2 Protein reconstitution baculoviruss, 26
In～28 DEG C of constant-temperature shaking incubators (with 180r/min), culture sampled microscopy after 7 days, calculated Cell viability.Virus liquid is with rotating speed
3000r/min is centrifuged 20 minutes, collects supernatant, and the swine fever virus e2 recombiant protein harvesting is carried out qualitative and quantitative analysis,
Result of the test is shown in Table 4.
Table 4 sf-9 cell inoculation recombinant baculovirus cell count result after 7 days
As shown in Table 4, through three batches of totally 9 verification experimental verifications, it is adjusted to sf-9 with insect cell medium and viral maintaining liquid respectively thin
Born of the same parents' density, 7 days after inoculation recombinant baculovirus, through t check analyses, under two kinds of different maintenance liquid system, living cells
There was no significant difference for density (p ＞ 0.05), and the significant difference (p ＜ 0.05) of Cell viability and results e2 protein content, with virus
After maintaining liquid adjustment cell density, system environment and nutrient, unit volume averagely can improve 4.08 μ g swine fever virus e2 restructuring eggs
White synthetic quantity.
2nd, high five cell suspension cultures expression e2 albumen by high five cell with sfx-insect serum-free elder brother
Worm cell culture fluid carries out suspension culture, when cell density reaches 3.0 × 106During individual/ml (cell survival rate is not less than 95%),
Cell equivalent sub-bottle is cultivated, uses insect cell medium and viral maintaining liquid adjustment cell density to 1.5 × 10 respectively6Individual/
Ml, inoculates equivalent swine fever virus e2 Protein reconstitution baculoviruss, in 26～28 DEG C of constant-temperature shaking incubators (with 180r/min)
Culture sampled microscopy after 4 days, calculated Cell viability.Virus liquid is centrifuged 20 minutes with rotating speed 3000r/min, collects supernatant, right
The swine fever virus e2 recombiant protein harvesting carries out qualitative and quantitative analysis, and result of the test is shown in Table 5.
Table 5 high five cell inoculation recombinant baculovirus cell count and protein quantification result after 4 days
As shown in Table 5, through three batches of totally 9 verification experimental verifications, it is adjusted to high with insect cell medium and viral maintaining liquid respectively
Five cell density, 4 days after inoculation recombinant baculovirus, through t check analyses, under two kinds of different maintenance liquid system,
There was no significant difference for viable cell density (p ＞ 0.05), the e2 protein content significant difference (p ＜ 0.05) of Cell viability and results,
After viral maintaining liquid adjustment cell density, system environment and nutrient, unit volume can improve 6.39 μ g swine fever virus e2 restructuring
Protein secretion.
To sum up, the insect cell maintaining liquid that the application is proposed, compared with conventional insect cell medium, not only can have
Effect improves the swine fever virus e2 recombiant protein content of insect cell-baculovirus expression, and largely reduces antigen
Production cost, is that the industrial amplification production of swine fever virus e2 Protein reconstitution baculoviruss inactivated vaccine is laid a good foundation, is elder brother
The optimization of worm cell-baculovirus expression system opens new approach.
Virus described herein maintains key component information in formula of liquid as follows: dextran sulfate sodium salt (sigma,
D1776), polyethers f-68 (sigma, p7061), l- L-Valine (sigma, v0513), glycine (sigma, g8790), dl- Soviet Union
Propylhomoserin (sigma, t1520), l- leucine (sigma, l8912), l- aspartic acid (sigma, a5474), l- agedoite
(sigma, a4159), glutamine dipeptide (sigma, a8185), sodium chloride (sigma, s5886), potassium chloride (sigma, p5405),
Potassium dihydrogen phosphate (sigma, p5655), disodium hydrogen phosphate (sigma, s5136), are more than cell culture level reagent.
Sf-9 cell line used herein, high five cell line are purchased from U.S.'s invitrogen life technology public affairs
Department；Sf900ii serum-free insect cell culture fluid is purchased from gibco company, article No.: 10902；Sfx-insect Insect cellculture
Liquid is purchased from hyclone company of the U.S., article No.: sh30278.02.
Method for cell count described herein: countstar automated cell calculating instrument.
Swine fever virus e2 recombiant protein content assaying method described herein is as follows:
1 materials and methods
1.1.1 the swine fever virus e2 albumen of albumen insect cell-rhabdovirus system expression, bovine serum albumin (thermo,
23209).
1.1.2 reagent swine fever positive serum (purchased from Chinese veterinary microorganism culture presevation administrative center), Radix Cochleariae officinalises peroxidating
The anti-pig of rabbit two of thing enzyme labelling resists (sigma, a5670), horseradish peroxidase substrate reagent box (vector, sk-4600), egg
White pre-dyed marker (fermentas, sm0671), sds-page gel reagent preparation box (solarbio, p1200), tris
Base (sigma, t1503), tween-20 (solarbio, t8220), Coomassie brilliant blue (amresco, c8430), pvdf film
(thermo, 88585), sds (sigma, s8010), skimmed milk, 1 × pbs, methanol, ethanol, acetic acid etc..
1.1.3 instrument, equipment, the small-sized Vertial electrophorestic tank of software bio-rad, bio-rad electrophresis apparatuses, bio-rad are small-sized wet
Formula transfer groove, horizontal decolorization swinging table, geldot-it310 gel imaging system (uvp) etc..Protein quantification software is
geldoc-it imaging system vision works.
1.2 method
1.2.1 sample treatment takes the swine fever virus e2 Protein reconstitution baculoviruss supernatant of expression on sf-9, high five cell
Liquid 1ml is to microcentrifugal tube.By taking out 5 × sample-loading buffer in -20 DEG C of refrigerators, take the supernatant of 80 μ l and 20 μ l 5 × on
After sample buffer mix homogeneously, after boiling 15 minutes in water-bath, put standby on ice.
1.2.2 protein standard liquid dilution take continuous 2 times dilution bovine serum albumin (bsa) titer 80 μ l (250,
125th, 62.5,31.25,15.625 μ g/ml) mix homogeneously with 20 μ l 5 × sample-loading buffers, after boiling 15 minutes in a water bath,
It is placed in standby on ice.
1.2.3 the electrophoresis of albumin glue with sds-page gel reagent preparation box prepare 2,12% glue, by albumen marker,
Sample and bsa protein standard liquid sequentially loading to film, with 150 volts of voltage, electrophoresis 80 minutes.
1.2.4, after transferring film electrophoresis finishes, a piece of film is used for transferring film, wet turn of 300ma 1 hour, so that protein is transferred to
On pvdf film, after the completion of take out film and put plastics and wash in box, add 5% skimmed milk of 25ml to close 30 minutes at room temperature.
1.2.5 an anti-reflective should outwell skimmed milk, is washed after three times with pbst, adds one in 1%pba (pbs+1%bsa)
Anti- swine fever positive serum (1 1000), puts room temperature reaction 1 hour, outwells one and resists, washes five each 20ml with pbst.
1.2.6 two anti-reflective should add the anti-pig of the rabbit of horseradish peroxidase-labeled two to resist (1 5000 dilution), shakes under room temperature
Shake reaction 1 hour, outwell two and resist, washed with pbst and develop the color after 6 times.
1.2.7 develop the color in darkroom, film is put in plate, first plus 1ml deionized water, then use horseradish peroxidase
Substrate reagent box develop the color.See specific band in about 48kda and then represent that expressed albumen can be identified by swine fever positive serum,
Insect cell-baculoviruss successful expression swine fever virus e2 albumen.
1.2.8 another glue of protein quantification is dyeed 30 minutes with coomassie brilliant blue staining liquid, then decolours, and becomes in gel
As imaging, preservation on instrument.According to Western blot result, determine swine fever virus e2 recombiant protein band, use gel imaging analysis system
System (uvp geldoc-it imaging system vision works), with bsa as reference, does standard curve quantitation swine fever
Viral e2 albumen.
Claims (9)
1. a kind of improve swine fever virus e2 expression of recombinant proteins amount insect cell maintaining liquid it is characterised in that: described insecticide is thin
Born of the same parents' maintaining liquid is by interpolation limiting amino acid, dextran sulfate sodium salt in 1 × pbs balance liquid-phosphate buffer and to gather
Ether f-68 forms.
2. insect cell maintaining liquid as claimed in claim 1 it is characterised in that: described 1 × pbs balance liquid-phosphate-buffered
Liquid is by sodium chloride 6000-10000mg/l, potassium chloride 100-300mg/l, disodium hydrogen phosphate 1000-2000mg/l, biphosphate
The concentrated hydrochloric acid composition of potassium 100-400mg/l and surplus, the concentrated hydrochloric acid of described surplus refers to above-mentioned 1 × pbs balance liquid-phosphate
The ph value of buffer is adjusted to 6.40 usage amount.
3. insect cell maintaining liquid as claimed in claim 2 it is characterised in that: described 1 × pbs balance liquid-phosphate-buffered
Liquid is by sodium chloride 8000mg/l, potassium chloride 200mg/l, disodium hydrogen phosphate 1440mg/l, potassium dihydrogen phosphate 240mg/l and surplus
Concentrated hydrochloric acid composition, the concentrated hydrochloric acid of described surplus refers to be adjusted to the ph value of above-mentioned 1 × pbs balance liquid-phosphate buffer
6.40 usage amount.
4. insect cell maintaining liquid as claimed in claim 1 it is characterised in that: described limiting amino acid refers to sweet ammonia
Acid, dl- threonine, l- L-Valine, l- leucine, l- aspartic acid, l- agedoite and glutamine dipeptide.
5. insect cell maintaining liquid as claimed in claim 4 it is characterised in that: described glycine 20-40mg/l, dl- revive ammonia
Sour 80-105mg/l, l- L-Valine 80-105mg/l, l- leucine 90-150mg/l, l- aspartic acid 20-40mg/l, l- Radix Asparagi
Amide 30-70mg/l, glutamine dipeptide 150-450mg/l.
6. insect cell maintaining liquid as claimed in claim 5 it is characterised in that: described glycine 30mg/l, dl- threonine
95mg/l, l- L-Valine 94mg/l, l- leucine 105mg/l, l- aspartic acid 30mg/l, l- agedoite 50mg/l and the third paddy
Dipeptides 300 mg/l.
7. insect cell maintaining liquid as claimed in claim 1 it is characterised in that: described sodium dextran sulfate 80-120mg/l；
Described polyethers f-68 is 800-1200mg/l.
8. insect cell maintaining liquid as claimed in claim 7 it is characterised in that: described sodium dextran sulfate 100mg/l；Described
Polyethers f-68 is 1000mg/l.
9. prepare claim 1 described in insect cell maintaining liquid method it is characterised in that: described compound method is: spends
Ionized water prepares 1 × pbs balance liquid-phosphate buffer, and Deca concentrated hydrochloric acid adjusts ph to after 6.40；To above-mentioned balance liquid-
In phosphate buffer add glycine, dl- threonine, l- L-Valine, l- leucine, l- aspartic acid, l- agedoite, third
Paddy dipeptides, sodium dextran sulfate and polyethers f-68, are filtered with 0.22 μm of sterilizing filter after fully dissolving；Finally, aseptic inspection
It is qualified to test, and osmotic pressure can be used as insect cell maintaining liquid in the solution of 350-380mosm/kg.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611113323.7A CN106367379B (en) | 2016-12-07 | 2016-12-07 | A kind of insect cell maintaining liquid and its preparation method improving swine fever virus E2 recombinant protein expression quantity |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611113323.7A CN106367379B (en) | 2016-12-07 | 2016-12-07 | A kind of insect cell maintaining liquid and its preparation method improving swine fever virus E2 recombinant protein expression quantity |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106367379A true CN106367379A (en) | 2017-02-01 |
| CN106367379B CN106367379B (en) | 2019-09-10 |
Family
ID=57891745
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611113323.7A Active CN106367379B (en) | 2016-12-07 | 2016-12-07 | A kind of insect cell maintaining liquid and its preparation method improving swine fever virus E2 recombinant protein expression quantity |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106367379B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593316A (en) * | 2015-02-02 | 2015-05-06 | 苏州市沃美生物技术有限公司 | Insect cell serum-free culture medium and application thereof |
| CN105242044A (en) * | 2015-11-23 | 2016-01-13 | 新疆天康畜牧生物技术股份有限公司 | Test method for hog cholera virus E2 protein quantification |
| CN105255811A (en) * | 2015-11-09 | 2016-01-20 | 内蒙古金源康生物工程有限公司 | Preparing method for serum-free and animal-source-free culture medium additive suitable for growth of insect cells |
| CN105255810A (en) * | 2015-11-09 | 2016-01-20 | 内蒙古金源康生物工程有限公司 | Serum-free and animal-source-free culture medium suitable for insect cell Sf-9 |
| CN105861416A (en) * | 2016-06-06 | 2016-08-17 | 西北民族大学 | Serum-free medium for culturing insect cells in full suspension mode and preparation method and application thereof |
| CN106148268A (en) * | 2015-04-13 | 2016-11-23 | 北京天信和生物科技有限公司 | A kind of serum-free insect cell culture medium and its preparation method and application |
-
2016
- 2016-12-07 CN CN201611113323.7A patent/CN106367379B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104593316A (en) * | 2015-02-02 | 2015-05-06 | 苏州市沃美生物技术有限公司 | Insect cell serum-free culture medium and application thereof |
| CN106148268A (en) * | 2015-04-13 | 2016-11-23 | 北京天信和生物科技有限公司 | A kind of serum-free insect cell culture medium and its preparation method and application |
| CN105255811A (en) * | 2015-11-09 | 2016-01-20 | 内蒙古金源康生物工程有限公司 | Preparing method for serum-free and animal-source-free culture medium additive suitable for growth of insect cells |
| CN105255810A (en) * | 2015-11-09 | 2016-01-20 | 内蒙古金源康生物工程有限公司 | Serum-free and animal-source-free culture medium suitable for insect cell Sf-9 |
| CN105242044A (en) * | 2015-11-23 | 2016-01-13 | 新疆天康畜牧生物技术股份有限公司 | Test method for hog cholera virus E2 protein quantification |
| CN105861416A (en) * | 2016-06-06 | 2016-08-17 | 西北民族大学 | Serum-free medium for culturing insect cells in full suspension mode and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 季新成等: "猪瘟病毒E2(gp55)基因在昆虫细胞中的表达", 《中国预防兽医学报》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106367379B (en) | 2019-09-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100389193C (en) | Method for safe continuous enclosed cell culture, virus production/ inactivation | |
| Staub | A century of research on mammalian male germ cell meiotic differentiation in vitro | |
| CN1147833A (en) | Hepatitis A virus culture process | |
| CN101736062A (en) | Method for preparing recombinant porcine alpha interferon standard substance | |
| CN107043738A (en) | A kind of porcine hepatocyte serum free medium and preparation method thereof | |
| CN104152403B (en) | A kind of method that establishing goose embryonic epithelium cell line and the goose embryonic epithelium cell line of foundation | |
| CN104178451B (en) | From milk, separate and cultivate method and the special culture media of cow mammary gland epithelial cells | |
| Ryan | Introduction to animal cell culture | |
| CN105154389A (en) | Low-serum protein-free culture medium suitable for PK-15 cell growth and preparation method of culture medium | |
| CN109609444A (en) | A kind of BHK21Cell non-serum culture medium | |
| CN106367379A (en) | Insect cell maintenance liquid for increasing expression amount of hog cholera virus E2 recombinant proteins and preparation method thereof | |
| CN109750058A (en) | A kind of research method of Candida albicans | |
| CN104630158A (en) | Method for producing porcine epizootic diarrhea CV777 strain virus by cultivating Vero cell in low serum | |
| CN105176915A (en) | Low-serum protein-free culture medium applicable to Marc-145 cell growth and preparation method thereof | |
| HU185997B (en) | Process for the cultivation of insect cells | |
| CN104630159A (en) | Method for producing hog cholera C-strain virus by culturing ST Cells in low serum | |
| CN104593334A (en) | Method for producing ring II type DBN-SX07 strain virus for pigs by seroculturing PK-15 cells | |
| CN109679900B (en) | Preparation method of avian influenza vaccine and product thereof | |
| CN105316282B (en) | A kind of acipenser dabryanus spermatogonium culture solution and application | |
| RU2353650C1 (en) | Line of cells of immature carp (cyprinus carpio) ico ovaries for virological research | |
| US4229541A (en) | In vitro cultivation of horseshoe crab amebocytes | |
| CN107406823A (en) | The peptide aqueogel of synthesis as extracellular matrix | |
| CN104651320A (en) | Method for producing porcine transmissible gastroenteritis Hua strain virus by low-serum culture of ST cells | |
| CN105176916A (en) | Low-serum protein-free culture medium applicable to Vero cell growth and preparation method thereof | |
| Pereira et al. | Optimized insect cell culture for the production of recombinant heterologous proteins and baculovirus particles |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20210421 Address after: Room 201, building 1, No.46, Weixing Road, phase II, Xinjiang Urumqi Economic Development Zone (Toutunhe District), Urumqi City, Xinjiang Uygur Autonomous Region 830000 Patentee after: Tiankang biopharmaceutical Co.,Ltd. Address before: 830011 No. 528 South Changchun Road, hi tech Zone, the Xinjiang Uygur Autonomous Region, Urumqi Patentee before: TECON BIOLOGICAL Co.,Ltd. |