CN102533721A - Method for improving transient expression of recombinant protein by abrupt change of osmotic pressure - Google Patents
Method for improving transient expression of recombinant protein by abrupt change of osmotic pressure Download PDFInfo
- Publication number
- CN102533721A CN102533721A CN2010106036367A CN201010603636A CN102533721A CN 102533721 A CN102533721 A CN 102533721A CN 2010106036367 A CN2010106036367 A CN 2010106036367A CN 201010603636 A CN201010603636 A CN 201010603636A CN 102533721 A CN102533721 A CN 102533721A
- Authority
- CN
- China
- Prior art keywords
- cell
- osmotic pressure
- transfection
- expression
- sudden change
- 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.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
A recombinant protein produced by a mammal cell has great commercial value. Methods for producing the recombinant protein by a transient transfection method have the advantages of quickness and generality, but have the defects of low transfection efficiency and protein expression, so the application of the methods is greatly limited. A method for improving the transient transfection efficiency of the mammal cell by the abrupt change of the osmotic pressure can improve the expression of the recombinant protein by 20 to 40 percent.
Description
Technical field
The present invention relates to biological technical field, be specially a kind of method that improves expression of recombinant proteins through the osmotic pressure sudden change.
Background technology
Along with molecular biology research deepens continuously, gene expression technique is greatly improved.Up to now, research and develop out multiple protokaryon and eukaryotic expression system in order to produce recombinant protein.The prokaryotic expression system mainly comprises escherichia expression system, subtilis expression system, streptomycete expression system.Eukaryotic expression system comprises yeast expression system, insect expression system, mammalian cell expression system and plant expression system.
The prokaryotic expression system that adopts the earliest; Also be to grasp the most sophisticated expression system at present; This technology mainly is to come transform bacteria with the carrier of cloning target gene fragment, obtains required target protein [Busuttil B E, Tumey K L through abduction delivering, purifying; Frauman A G.Protein Expression and Purification, 2001; 23 (3): 369-373.Leandro P, Lechner M C, Almerda H, etal.Molecular Genetics and Metabolism, 2001; 73 (2): 173-17].Because the microbial culture in the prokaryotic expression system is simple to operate, growth and breeding is fast, cheap, the exogenous genes products expression level is high, genetic background and expression characterization are known etc. factor makes this system become one of most popular heterologous protein expression system.But prokaryotic expression system also has a lot of deficiencies, and its main drawback has: 1. do not have the function of eukaryotic transcription post-treatment, can not carry out the montage of mRNA, can not express the genomic gene of eucaryon so can only express cDNA; 2. the function that does not have the eukaryotic translation post-treatment is expressed the protein that produces, and can not carry out modifications such as glycosylation, phosphorylation, and it is folding to be difficult to form correct disulfide linkage pairing and space conformation, thereby the protein biology poor activity that produces; 3. expressed protein is gathered into inclusion body [Kane J F, Hartiey D L.Tibtech, 1988 through regular meeting in bacterium; 6:95-102], cause product renaturation difficulty, activity is not good.
System is modified in the processing that eukaryotic expression system has after the translation, and the foreign protein of expression more approaches natural protein.Therefore, utilizing eukaryotic expression system to express target protein more and more comes into one's own.At present, eukaryotic expression system commonly used has mammalian cell expression system, yeast expression system and insect cell expression system in the genetically engineered research.Mammalian cell expression system can instruct proteinic correct folding; Multiple translation post-treatment functions such as complicated N type glycosylation and O type glycosylation accurately are provided, and its expression product is approaching natural higher organism protein molecule most aspect molecular structure, physicochemical property and the biological function, outclass eukaryotic expression system [Chen P such as prokaryotic expression system and yeast, insect cell aspect active; Hutter D; Liu P, et al.Protein ExprPurif, 2002; 24 (3); 481-488].At present, mammalian cell expression system has become several genes engineering production of medicine platform, and in the discovery of new gene, the research of proteinic 26S Proteasome Structure and Function, has play a part very important.
What use always in the mammalian cell expression system is the stable transfection system; Its principle is that foreign DNA is incorporated in the karyomit(e) of host cell; Through some selected markers; Tathagata p-Beta Aminopropionaldehyde, hygromycin B phosphotransferase (HPH), thymidine kinase repeated screenings such as (TK) obtain the isologous cell line of stable transfection.Though stable expression system is the gold standard of biological production system.Yet transfection wherein and cell screening process need labor time and manpower [Geisso S, Kocher HP. [J] .Methods Enzymol, 1999,306:19].And, be badly in need of thousands of hundreds of target protein and carry out research and analysis along with engineered develop rapidly.This just requires in the short as far as possible time, to obtain a large amount of expression of recombinant proteins products, so transient gene expression obtained very big development as a kind of heterologous gene expression system fast, easily, and its foreign gene is present on the free carrier after getting into recipient cell, does not integrate mutually with host cell chromosome; Its number in cell reduces along with the division of cell usually, so protein expression can only keep several days to tens days time, stablizes the gene expression method of strain with respect to making up, and transient gene (protein) expression method just can obtain target protein [Cullen, B.R. (1987) .Methods Enzymol. in several weeks after obtaining gene; 152,684-704.Blasey, H.D., Aubry, J.-P.; Mazzei, G.and Bernard, A. (1996) .Cytotechnology, 18,183-192.Cachianes; G., Ho, C., Weber; R.F., Williams, S.R., Goeddel; D.V.and Leung, D.W. (1993) .Biotechniques, 15,255-259.].
At present, the method for a variety of transmission DNAs is arranged, except that virus vector, mainly contain the calcium phosphate coprecipitation method, DEAE-glucose method, liposome transfection method, electroporation and particle gun blast technique.Electroporation is under the effect of high-voltage electric field, and the provisional formation micropore that breaks takes place cytolemma, can make macromole and small molecules get into cell interior from the external world, thereby or oppositely flow out the transfection that cell is realized DNA; Gene gun technology is the very high initial velocity of small gold grain (or tungsten powder) that is enclosed with DNA through offering, and makes its penetrate plant cell wall and reaches the purpose that shifts foreign DNA.The principle of calcium phosphate method transfection is to mix the plasmid that contains foreign DNA with calcium phosphate transfection liquid, joins by in the cells transfected culture environment, under the media of calcium phosphate transfection liquid, makes foreign DNA get into cell.DEAE-VISOSE mediated method transfection principle is that the electronegative phosphoric acid skeleton of DEAE-VISOSE and the nucleic acid of positively charged interacts the mixture that forms by cell endocytic.The mechanism that lipid mediated gene transfer is possible is that cationic-liposome and electronegative gene borrow electrostatic interaction to form the liposome gene mixture; This mixture is because of the superfluous positive charge positively charged of cationic-liposome; Be adsorbed in electronegative cell surface by electrostatic interaction, again through getting in the cell with cytolemma fusion or endocytosis.Various transfection methods, DNA gets into the needed asynchronism(-nization) of cell, and by means of the transfection method that transfection reagent carries out, the process of DNA entering cell can continue the time of several hrs usually.
Though the foreign gene transient expression has very big advantage in speed with above the versatility, and along with the use of cheap transfection reagents such as calcium phosphate and PEI, this technology has obtained very big application [Boussif, O., Lezoualc ' h; F., Zanta, M.A., Mergny; M.D., Scherman, D., Demeneix; B.and Behr, J.P. (1995) Proc.Natl Acad.Sci.USA, 92,7297-7301].But it has the shortcoming that transfection efficiency is low, expressing quantity is low and causes its application to receive very big restriction.
Some improves transient transfection Study on Efficiency report before, such as: select suitable transfection reagent; Optimize the ratio of transfection reagent and DNA; Design is fit to the carrier that transient transfection is expressed; Express with some growth factor cotransfections; Add proteolysate during cell cultures after the transfection; PH through the control substratum; Conversion and control through temperature improves the Recombinant Protein Expression amount; In transfection process, cellular exposure is increased the percentage ratio of infected cell in Sodium propanecarboxylate solution, induced protein synthetic; Use 2-aminopurine to improve Recombinant Protein Expression amount [DUROCHER, Y.et al., Anal Biochem, 2000.284 (2): p.316-26.BALDI; L, et al., Biotechnol Prog, 2005.21 (1): p.148-53.MEISSNER; P.et al., Biotechnol Bioeng, 2001.75 (2): p.197-203.STETTLER, M; Et al., Biotechnol Bioeng, 2006.FUSSENEGGER, M; Et al., Nat Biotechnol, 1998.16 (5): p.468-72].Improve the high-density growth and the albumen high expression level of cell after transfection efficiency and the transfection of cell through top method.U.S. Pat 2008/0145893 gets up to use with these factors combine, has reached a higher expression and transfection efficiency, but its complex process, agents useful for same is expensive, is not suitable for large-scale promotion and uses.
The research of the influence of osmotic pressure pair cell expressing protein also has more report, and the research of Ryu etc. shows that high osmotic pressure can make the Chinese hamster ovary celI generation G1 phase block, thereby improves output [Ryu J S, the Lee M S of target protein; Lee, G M, [J] .Biotechnol Prog; 2001,17 (6): 993～999.] Kim etc. increases Chinese hamster ovary celI foreign protein output with the method that improves osmotic pressure, adopts two stage training strategies: make cell grow to proper density fast with standard medium (294mmol/L); Use high osmotic pressure (522mmol/L) substratum replacement standard medium then, make the output of target protein improve 161% [Kim M S, Kim N S; Sung Y H, etal, [J]; InVitro Cell Dev Biolanim, 2002,38 (6): 314-319].Sun etc. have comparatively systematically studied high osmotic pressure to the influence of transcribing, translating and translate processes such as post-treatment of Tegeline in mouse hybridoma, find that high osmotic pressure can improve the speed of proteic translation skill and translation post-treatment, cell volume are increased and antibody expression level raising [Sun Z; Zhou R, Liang S, etal; [J]; Biotechnol Prog, 2004,20 (2); 576-589].
In these reports, all be to increase protein expression level through the osmotic pressure of cell after the adjustment transfection, we do not find in the transfection process to improve through the osmotic pressure that changes cell the relevant report of transfection efficiency.This patent provides a kind of osmotic pressure through the nutrient solution of cell before and after the change transfection to improve the method for recombinant protein transient expression, can on former basis, make the Recombinant Protein Expression amount improve more than 20%.
Summary of the invention
The recombinant protein of producing with mammal cell line system has very high commercial value, however use at present maximum be that the structure of labor manpower and time is stablized the method for strain.Method based on the foreign gene transient expression has very big advantage in speed with above the versatility, and has special advantages expressing cytotoxicity specifically to be arranged or influence on the fissional albumen.Along with the use of transfection reagents such as liposome, calcium phosphate and PEI, this technology has obtained very big application.But with respect to stablizing the strain expression system, transient transfection is produced recombinant protein and is had the shortcoming that transfection efficiency is low, expressing quantity is low, makes the application of this method receive bigger restriction.
This patent provides a kind of method through cell environment osmotic pressure of living in before and after the sudden change transfection to improve the transfection efficiency of mammalian cell and then improve the Recombinant Protein Expression amount.Can on former basis, make the Recombinant Protein Expression amount improve more than 20%.
The present invention is reduced to a scope of forcing down than the suitable infiltration of cell growth with the osmotic pressure of cell environment of living in before transfection; Make it be easier to introduce foreign DNA; Thereby improve the transfection efficiency of cell, cell is got back to the osmotic pressure cultivation of suitable growth after the transfection, keeps the growth and the expression environment of the best of cell as far as possible; The murder by poisoning that reduces the osmotic pressure sudden change and introduce the plasmid pair cell, thus make the cell high-density growth realize the high expression level of foreign DNA proteins encoded.
Osmotic pressure mutational range of the present invention is to change with the different transfection method of different cells with amplitude, and those of ordinary skill in the art can confirm the scope and the amplitude of osmotic pressure sudden change easily.In a preferred embodiment of the invention; Select for use HEK 293 cells to carry out the osmotic pressure sudden change; Before carrying out transfection, the 265mOsm/kg of its residing osmotic pressure from suitable growth suddenlyd change to 230-175mOsm/kg; After continuing for some time, get back to that culturing cell carries out protein expression under the osmotic pressure of 265mOsm/kg.Expressing quantity and Qp are significantly improved.
The time of origin of the sudden change of the osmotic pressure among the present invention can be certain time before foreign DNA adds cell culture fluid, in the time of also can being foreign DNA adding cell culture fluid.Those of ordinary skill in the art can confirm the time of origin of the osmotic pressure sudden change of different transfection methods and different cells easily.In the preferred embodiment of the present invention; Time of origin to the sudden change of the osmotic pressure of HEK 293 cells is optimized; Beginning sudden change when determining 0-1 hour before DNA-transfection reagent mixture adds cell culture fluid is reasonable scope; The preferable growth that this moment, sudden change can keep cell can improve the transfection efficiency of cell again, thereby obtain the high expression level of foreign DNA proteins encoded.Expressing quantity improves 22.7%-31.1%.
The termination time of the sudden change of the osmotic pressure among the present invention can be certain time after foreign DNA adds cell culture fluid, in the time of also can being foreign DNA adding cell culture fluid.Those of ordinary skill in the art can confirm the termination time of the osmotic pressure sudden change of different transfection methods and different cells easily.In the preferred embodiment of the present invention; Termination time to the sudden change of the osmotic pressure of HEK 293 cells is optimized; Finishing sudden change when determining 0-4 hour after DNA-transfection reagent mixture adds cell culture fluid is reasonable scope; The preferable growth that this moment, sudden change can keep cell can improve the transfection efficiency of cell again, thereby obtain the high expression level of foreign DNA proteins encoded.The highest one group expressing quantity improves 34.9%.
Cell among the present invention can select the mode of various raising protein expressions to cultivate through after the transfection, expresses strategies such as temperature such as feeding culture, adding butyrates, reduction.The method of expression of recombinant proteins can make up use in the method for the transient transfection among the present invention and all raising cell, does not receive any restriction.
The carrier of using among the present invention can be a commercialization carrier arbitrarily, and those of ordinary skill in the art can both select suitable carriers to carry out plasmid construction.
Method of the present invention is applicable to the transient transfection of various mammalian cells, in the specific embodiment of the present invention, optimizes with HEK 293 cells, has obtained effect preferably.
Method of the present invention is applicable to suspension cell or attached cell, in specific embodiment of the present invention, carries out the osmotic pressure sudden change with adherent culture and suspension culture respectively, all obtains effect preferably.
Method of the present invention is applicable in the substratum of various suitable corresponding transfection methods.
" transfection " among the present invention is meant the process of foreign DNA being introduced cell, claims then that when foreign DNA gets into cell interior through cytolemma this cell is by transfection.Many rotaring dyeing technologies that those of ordinary skill in the art knows can both be introduced cell such as plasmid vector and other nucleic acid molecule with one or more nucleic acid synthetics.Method of the present invention is applicable to the method for various transient transfections, such as coprecipitation of calcium phosphate method, liposome transfection, the tree-shaped polymkeric substance transfection of activatory, the transfection of polyamines reagent.Various transfection methods can carry out under the optimized conditions at it separately.
" albumen " is meant by a large amount as far as possible and expresses and the product albumen of results among the present invention, and it can be any significant albumen, antibody, antibody fragment or polypeptide.An EGFR albumen and any IgG antibody have been used in the specific embodiment of the present invention.Product collection and be used for purifying to obtain proteic downstream processing technology from culture be the usual manner that those of ordinary skills know includes but not limited to: technology such as centrifugal, ultrafiltration, ion exchange chromatography, affinity purification.
Change the osmotic pressure of cell cultures " osmotic pressure sudden change " the opportunity that is meant among the present invention, return to the process of osmotic pressure before changing after the lasting specific time again specific.
" transient expression " among the present invention be meant foreign DNA introduced mammalian cell, but the foreign DNA unconformability is on host cell chromosome, but transcribed, translate, finally give expression to the foreign DNA encoded protein with the free form.
The present invention is as simple and effectively improve the method for recombinant protein transient expression; It has very large commercial value, can replace traditional structure cytotostatic strain expression system to produce such as cytotoxic albumen, block fissional albumen.Can obtain recombinant protein fast, life science and drug development are had great supporting role.
" expression rate of unit time unit cell " " cell expressing rate " " Qp " among the present invention is meant 24 hour the expression amount of target recombinant protein in individual cells.
" expression amount " among the present invention is meant the concentration of target protein, and this concentration can be that the Elisa detection method gets or the result through drawing behind the protein purification of downstream.
Description of drawings
Fig. 1. the growing state of HEK 293 cells after transfection of transfection under the different osmotic condition.Suddenling change, cells transfected does not have too much influence to its growth under the 213mOsm/kg-281mOsm/kg osmotic pressure, but suddenlys change to being lower than 213mOsm/kg and being higher than cells transfected under the 281mOsm/kg osmotic pressure, and the growth after the transfection is relatively poor.
Fig. 2. HEK 293 cells of transfection under the different osmotic condition, the expression of albumen EGFR after the transfection.Expression amount is the highest at the cells transfected expression amount that suddenlys change under the 190mOsm/kg osmotic pressure.
Fig. 3. HEK 293 cells of transfection under the different osmotic condition, the variation of the expression rate after the transfection (Qp).The reduction of the osmotic pressure that suddenlys change during along with transfection, Qp is the highest at the cells transfected Qp that suddenlys change under the 190mOsm/kg osmotic pressure.
Fig. 4. the influence of the growth of the different time of osmotic pressure sudden change beginning after to HEK 293 cell transfectings.The cell growth is good more closely more with the time of the time gap DNA adding cell culture fluid of osmotic pressure mutation initiation, adds cell at distance B NA and begins sudden change in the time of 1-0 hour, and is consistent with the cell growth of the control group that does not carry out the osmotic pressure sudden change.
Fig. 5. the different time of osmotic pressure sudden change beginning to HEK 293 cell transfectings after the expression of albumen EGFR.Protein expression the best appears at osmotic pressure distance B NA adding cell and begins sudden change in the time of 1-0 hour, and this moment, expressing quantity increased 22.7%-31.3%.
Fig. 6. the different time of osmotic pressure sudden change beginning to HEK 293 cell transfectings after the influence of expression rate (Qp).Qp is low more closely more with the time of the time gap foreign DNA adding cell culture fluid of osmotic pressure mutation initiation, also shows the prolongation of the time of osmotic pressure sudden change, helps the raising of transfection efficiency.
Fig. 7. the influence of the growth of the different time that osmotic pressure sudden change continues after to HEK 293 cell transfectings.Cell growth was influential when the osmotic pressure sudden change lasted till 4 hours behind the foreign DNA adding cell culture fluid, and the time before 4 hours does not all have tangible influence.
Fig. 8. the different time that osmotic pressure sudden change continues to HEK 293 cell transfectings after the expression of albumen EGFR.In the mutation time in scope of experiment, expression amount is improved, and the highest one group expressing quantity improves 34.9%.
Fig. 9. the different time that osmotic pressure sudden change continues to HEK 293 cell transfectings after the situation of protein expression rate (Qp).All conditions in scope of experiment, the Qp of osmotic pressure sudden change is all than the raising by a relatively large margin that has that does not suddenly change, and increase rate is at 30.5%-47.3%.
Figure 10. the influence of osmotic pressure sudden change antagonist expression amount.Osmotic pressure sudden change group improves 23.3% than control group expression amount.
Figure 11. the osmotic pressure sudden change is expressed the proteic influence of EGFR to HEK 293 attached cells.The expressing quantity of osmotic pressure sudden change group improves 31% than the expression amount of normal control group.
Embodiment
Material and method
Material
The proteic plasmid of EGFR, the IgG antibody plasmid of high efficiency transfection reagent Sinofection, band Fc label all come from Beijing justice and stick up Divine Land Bioisystech Co., Ltd (China)
Cell cultures
HEKC HEK 293 (ATCC) attached cell is cultivated in the T75 Tissue Culture Flask with the DMEM+10%FBS substratum, treats that abundance is about 90%, uses the trysinization passage cell.Put 37 ℃, cultivate in the 8%CO2 incubator.Before the transfection, get abundance and be about 90% cell, the trysinization counting.With the substratum of DMEM+10%FBS with cell dilution to 0.9 * 10
6Cells/ml gets 0.4ml and puts in the hole of 24 orifice plates, puts into 37 ℃ after cell is shaken up, and cultivates transfection after 3-5 hour in the 8%CO2 incubator.After the transfection 24 orifice plates are put 37 ℃, 8%CO
2Continue to cultivate in the incubator, after the transfection 72 hours the time sampling detect protein concentration.
HEKC HEK 293 tames in SBI-293TM (Beijing justice is stuck up Divine Land Bioisystech Co., Ltd, the China) substratum through suspending, and cell went down to posterity in per three days, and the initial density that goes down to posterity is 0.3～0.6 * 10
6Cells/ml, cell cultures is carried out in shaking bottle, and working volume is 15～35% of a TV, shaking speed: 100～130rpm, temperature: 36.5 ℃, carbonic acid gas: 5%.Before the transfection, get the cell that is in logarithmic phase, the centrifugal required cell density that goes down to posterity is put in the shaking table, and 100～130rpm cultivates down for 36.5 ℃, and 100ml shakes every bottle of culture of putting 30ml in the bottle, carries out transfection in the time of design then.Cell after the transfection continues at 100～130rpm; 36.5 cultivate in ℃ shaking table, and the mixing of the glucose of every 200mM L-glutaminate that added 1.2ml at a distance from 48 hours of the beginnings in 24 hours after adding DNA-transfection reagent mixture and 400g/L adds feed liquid and cultivates with the raising cell and grow and Recombinant Protein Expression.72 hours and 144 hours take a sample detection cell density and protein concentrations after the transfection.
Transfection
Cell suspension with transfection 30ml is an example: get in the NaCl solution of 150mM that 30ug DNA is diluted to 1.5ml and mix; Other gets in the NaCl solution of 150mM that 90ul high efficiency transfection reagent Sinofection is diluted to 1.5ml and mixes; Then dna solution and transfection reagent solution are mixed; Hatch 10-20min under the room temperature, process DNA-transfection reagent mixed solution.Dropwise add then and want in the cells transfected suspension-s.Transfection in the orifice plate, the amount of its used DNA and transfection reagent and NaCl solution reduces in proportion.
Cell counting
Come counting cells density with hematimeter; Judge the motility rate of cell with the trypan blue staining.
Protein Detection
Adopt the content of IgG antibody (albumen of Fc label) in double antibodies sandwich enzyme linked immunological (Elisa) test sample.To resist the specific antibody and the solid phase carrier of human IgG (Fc) to connect, form insolubilized antibody.Unconjugated antibody and impurity are removed in washing.Add standard substance and testing sample, the human IgG in sample and the standard substance (Fc) combines with insolubilized antibody, forms the solid phase antigen antibody complex; Then, add anti-human IgG (Fc) antibody of enzyme labelling, insulation reaction, the antigen on the solid-phase immunity mixture combines with enzyme labelled antibody.Receive the amount of human IgG in the sample (Fc) relevant in the enzyme amount that have on the solid phase carrier this moment and the sample.Add the substrate colour developing.Substrate for enzymatic activity on the solid phase becomes coloured product.Through colorimetric, predict the amount of the albumen (or human IgG antibody) of Fc label in the sample.
Above method is the routine operation method of biological technical field, by those of ordinary skill in the art is known.
Embodiment 1: the osmotic pressure sudden change is to the influence of expression of recombinant proteins
The influence that the sudden change pair cell transient transfection process of the osmotic pressure of cell environment of living in produces when analyzing transfection; Test with the design of HEK 293 cells is following: through the concentration of adjustment NaCl in medium; Obtain the substratum of a series of osmotic pressure, be respectively 175,190,213,230,265,281,306,330mOsm/kg, and in transfection preceding 1 hour; Get the healthy centrifugal liquid that changes of seed cell in the substratum of different osmotic pressure, 3 parallel appearance of each osmotic pressure condition.And keeping cell density is 1.0 * 10
6Cells/ml is put 100～130rpm, and 36.5 ℃, 5%CO
2Cultivate in the shaking table, carry out transfection after 1 hour.All cells DNA-transfection reagent mixture addition sequence at random, the joining day differs and is no more than 5 minutes.DNA-transfection reagent mixture added behind the cell culture fluid 1 hour, and the centrifugal liquid that changes is got back in the substratum of osmotic pressure 265mOsm/kg cell and cultivated.And the mixing of the glucose of every 200mM L-glutaminate that added 1.2ml at a distance from 48 hours of the beginnings in 24 hours after adding DNA-transfection reagent mixture and 400g/L adds feed liquid and cultivates, and grows and Recombinant Protein Expression with the raising cell.Took a sample in 72 hours and 144 hours and detect cell density and protein concentration.
Cell growth and expression of results are shown in Fig. 1-3; The transfection efficiency influence of the osmotic pressure pair cell during transfection is bigger, is in particular in: in scope of experiment, along with the reduction of osmotic pressure; Qp increases, and cell begins to occur tangible reduction when being grown in 190mOsm/kg.Protein expression has optimum value in the 190-213mOsm/kg scope, expression amount is than improving 29% transfection the time under the 265mOsm/kg osmotic pressure, and Qp improves 25.7-41.8%.
Embodiment 2:HEK 293 Premeabilisation of cells are pressed confirming of mutation initiation time
The time of the osmotic pressure sudden change of HEK 293 cells is confirmed in the following experiment of design: get the healthy cell that is in logarithmic phase; 4hr before transfection, 2hr, 1hr, 0.5hr, 0hr centrifugal treating respectively carry out transfection then in the substratum of 190mOsm/kg osmotic pressure.All cells DNA-transfection reagent mixture addition sequence at random, the joining day differs and is no more than 5 minutes.Centrifugal changing in the substratum that liquid is 265mOsm/kg to osmotic pressure cultivated when DNA-transfection reagent mixture added behind the cell culture fluid 1 hour.And the mixing of the glucose of every 200mM L-glutaminate that added 1.2ml at a distance from 48 hours of the beginnings in 24 hours after adding DNA-transfection reagent mixture and 400g/L adds feed liquid and cultivates, and grows and Recombinant Protein Expression with the raising cell.72 hours and 144 hours take a sample detection cell density and protein concentrations behind the DNA-transfection reagent mixture adding cell culture fluid.
Experimental result is shown in Fig. 4-6; Different time before the transfection carries out the osmotic pressure sudden change; Qp increases with the prolongation of the time of sudden change in scope of experiment; But cell growth increase and variation in time, the combined effect of two kinds of trend causes before transfection, suddenling change in 1-0.5 hour preferable expressing quantity is arranged, and this moment, expressing quantity increased 22.7%-31.3%.
Embodiment 3:HEK 293 Premeabilisation of cells are pressed confirming of sudden change time length
The time length of cell osmotic pressure sudden change in transfection process is confirmed in the following experiment of design: get the healthy cell that is in logarithmic phase; In transfection preceding 0.5 hour; Centrifugal treating is in the substratum of 190mOsm/kg osmotic pressure respectively; 0.5 carry out transfection after hour, all cells DNA-transfection reagent mixture addition sequence at random, the joining day differs and is no more than 5 minutes.And 0 hour after DNA-transfection reagent mixture adds the cell cultures nutrient solution respectively, 0.5 hour, 1 hour, 2 hours, the centrifugal liquid that changes of the osmotic pressure of cell environment of living in adjusted to 265mOsm/kg and continue to cultivate in 4 hours.And the mixing of the glucose of every 200mM L-glutaminate that added 1.2ml at a distance from 48 hours of the beginnings in 24 hours after adding DNA-transfection reagent mixture and 400g/L adds feed liquid and cultivates, and grows and Recombinant Protein Expression with the raising cell.72 hours and 144 hours take a sample detection cell density and protein concentrations behind the DNA-transfection reagent mixture adding cell culture fluid.
Experimental result is shown in Fig. 7-9, and cell is grown in osmotic pressure sudden change and lasts till obvious variation when adding behind the DNA-transfection reagent mixture 4 hours.All conditions in scope of experiment, the Qp of osmotic pressure sudden change is all than the raising by a relatively large margin that has that does not suddenly change, and increase rate is at 30.5%-47.3%, and the expression amount of EGFR also is improved, and increase rate is at 6.2%-39.6%.
Embodiment 4: the influence of the expression of osmotic pressure sudden change antagonist
The influence of osmotic pressure sudden change to HEK 293 cell transient transfections production antibody confirmed in the following experiment of design: get the healthy cell that is in logarithmic phase; At transfection eccentric visual cell in the time of preceding 0.5 hour; Be resuspended in the substratum of 190mOsm/kg osmotic pressure; Be distributed into three bottles, carry out transfection after 0.5 hour, and after DNA-transfection reagent mixture adds cell culture fluid 1 hour adjusts to the centrifugal liquid that changes of the osmotic pressure of cell environment of living in 265mOsm/kg and continues to cultivate.Three parallel appearance of another group, the centrifugal liquid that changes does not carry out the osmotic pressure sudden change as control group in the substratum of 265mOsm/kg, common transfection and cultivation.All cells DNA-transfection reagent mixture addition sequence at random, the joining day differs and is no more than 5 minutes.Two groups all the mixing of the glucose of the every 200mM L-glutaminate that added 1.2ml at a distance from 48 hours of the beginnings in 24 hours after adding DNA-transfection reagent mixture and 400g/L add feed liquid and cultivate, grow and Recombinant Protein Expression with the raising cell.72 hours and 144 hours take a sample detection cell density and ACs behind the DNA-transfection reagent mixture adding cell culture fluid.
Experimental result is shown in figure 10, and osmotic pressure sudden change group improves 23.3% than control group expression amount.
Embodiment 5.: the application of osmotic pressure sudden change in attached cell
Investigate the influence of osmotic pressure sudden change pair cell expression of recombinant proteins under the adherent situation with HEKC HEK 293 attached cells.Handle two groups of cells well, three every group parallel appearance, preceding 0.5 hour in transfection; One group of cell culture fluid wherein is replaced by the substratum that osmotic pressure is 190mOsm/kg; Another group is not carried out the osmotic pressure sudden change as contrast, only is replaced by the substratum of 265mOsm/kg osmotic pressure, carries out transfection after 0.5 hour; All cells DNA-transfection reagent mixture addition sequence at random, the joining day differs and is no more than 5 minutes.DNA-transfection reagent mixture added behind the cell 1 hour, and substratum is replaced by the substratum of 265mOsm/kg osmotic pressure, put in 36.5 ℃ the incubator to cultivate.Sampling detected the concentration of EGFR when DNA-transfection reagent mixture added behind the cell culture fluid 72 hours.
The result is shown in figure 11: the expressing quantity of osmotic pressure sudden change group improves 31% than the expression amount of normal control group.
Claims (7)
1. utilize osmotic pressure to suddenly change and improve the method for recombinant protein transient expression efficient in mammalian cell, it is characterized in that: before cell transfecting, reduce the osmotic pressure of cell culture fluid, again osmotic pressure is returned to after the transfection and reduce preceding level.
2. the reduction of osmotic pressure described in the claim 1 scope is 10-100mOsm/kg.
3. the reduction of osmotic pressure described in the claim 2 scope is 30-70mOsm/kg.
4. the reduction of the osmotic pressure described in the claim 1 time of origin is that foreign DNA added cell culture fluid 0-1 hour before.
5. the osmotic pressure described in the claim 1 is that foreign DNA added after the cell culture fluid 0-4 hour time of recovery.
6. the mammalian cell described in the claim 1 is HEKC (HEK 293) or Chinese hamster ovary cell (CHO).
7. the recombinant protein described in the claim 1 is albumen, antibody, antibody fragment or polypeptide.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106036367A CN102533721A (en) | 2010-12-24 | 2010-12-24 | Method for improving transient expression of recombinant protein by abrupt change of osmotic pressure |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010106036367A CN102533721A (en) | 2010-12-24 | 2010-12-24 | Method for improving transient expression of recombinant protein by abrupt change of osmotic pressure |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102533721A true CN102533721A (en) | 2012-07-04 |
Family
ID=46341754
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010106036367A Pending CN102533721A (en) | 2010-12-24 | 2010-12-24 | Method for improving transient expression of recombinant protein by abrupt change of osmotic pressure |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102533721A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105779501A (en) * | 2014-12-26 | 2016-07-20 | 上海药明康德新药开发有限公司 | Method for carrying out transient expression production on recombinant protein by adopting CHO (Chinese Hamster Ovary) host cells |
| CN106029871A (en) * | 2014-01-29 | 2016-10-12 | 株式会社Lg生命科学 | Method for controlling galactosylation of recombinant protein through optimization of culture medium |
| CN110551754A (en) * | 2019-07-26 | 2019-12-10 | 佛山汉腾生物科技有限公司 | Transient transfection method of CHO cell and recombinant CHO cell |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718734A (en) * | 2005-09-09 | 2006-01-11 | 中山大学附属第一医院 | Peritoneal transgene method |
| CN101603053A (en) * | 2008-06-10 | 2009-12-16 | 重庆宗申军辉生物技术有限公司 | Be used for mosaic type adenoviral vector of transfecting pig skin and uses thereof |
| CN101613710A (en) * | 2008-06-24 | 2009-12-30 | 重庆宗申军辉生物技术有限公司 | A kind of method of utilizing hexadimethrine bromide to improve transfection efficiency of gene transfected pigskin |
-
2010
- 2010-12-24 CN CN2010106036367A patent/CN102533721A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718734A (en) * | 2005-09-09 | 2006-01-11 | 中山大学附属第一医院 | Peritoneal transgene method |
| CN101603053A (en) * | 2008-06-10 | 2009-12-16 | 重庆宗申军辉生物技术有限公司 | Be used for mosaic type adenoviral vector of transfecting pig skin and uses thereof |
| CN101613710A (en) * | 2008-06-24 | 2009-12-30 | 重庆宗申军辉生物技术有限公司 | A kind of method of utilizing hexadimethrine bromide to improve transfection efficiency of gene transfected pigskin |
Non-Patent Citations (1)
| Title |
|---|
| 董菊子: "电转染效率影响因素的探讨", 《生物技术通讯》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106029871A (en) * | 2014-01-29 | 2016-10-12 | 株式会社Lg生命科学 | Method for controlling galactosylation of recombinant protein through optimization of culture medium |
| CN105779501A (en) * | 2014-12-26 | 2016-07-20 | 上海药明康德新药开发有限公司 | Method for carrying out transient expression production on recombinant protein by adopting CHO (Chinese Hamster Ovary) host cells |
| CN110551754A (en) * | 2019-07-26 | 2019-12-10 | 佛山汉腾生物科技有限公司 | Transient transfection method of CHO cell and recombinant CHO cell |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103320390B (en) | Large-scale mammal engineering cell cultural method | |
| CN107523588B (en) | Tetracycline lentivirus induced expression vector and establishment method and application thereof | |
| CN114085841B (en) | Site for stably expressing protein in CHO cell gene NW _003614092.1 and application thereof | |
| CN107201352A (en) | A kind of beta galactosidase combination mutant with high transglycosylation and its preparation method and application | |
| CN112522173A (en) | Engineering bacterium for producing heterologous alkaline protease and construction method thereof | |
| BR112020005217A2 (en) | methods for genetic engineering of kluyveromyces host cells | |
| CN116286900A (en) | Acetate osmotic enzyme A gene RkAcpa and application thereof | |
| CN102533721A (en) | Method for improving transient expression of recombinant protein by abrupt change of osmotic pressure | |
| CN105238816A (en) | Expression vector | |
| CN113969284B (en) | Site for stably expressing protein in CHO cell gene NW _003614889.1 and application thereof | |
| CN110499274A (en) | A kind of genetic engineering Rhodococcus sp and its construction method and application | |
| CN102533722B (en) | Method for improving transient expression of recombinant proteins in mammalian cells through temperature jump | |
| TW201738263A (en) | A preparation method of recombinant human granulocyte colony stimulating factor | |
| JP6469371B2 (en) | A method for expressing a plurality of foreign genes in an embryoid body composed of induced pluripotent stem cells (iPS cells) | |
| CN112592898B (en) | Reprogrammed NK feeder cells and preparation method and application thereof | |
| CN112626028B (en) | Engineering cell for activating NK-like cells and preparation method and application thereof | |
| CN109402096B (en) | AID enzyme mutant and application thereof | |
| CN110669788B (en) | Porphyridium chloroplast expression system and application thereof | |
| Lee et al. | Industrial application and genetic engineering of plant cell cultures | |
| KR101660805B1 (en) | Transformant transformed by Glut-1 gene and Methods for culturing the Same | |
| CN108424459A (en) | The fusion protein and its preparation method and application of human serum albumins and people's saltant type hepatocyte growth factor | |
| CN116769832B (en) | Transient transfection method of mammalian cells | |
| CN113025545B (en) | Blue algae engineering bacterium capable of efficiently fixing nitrogen and preparation method and application thereof | |
| CN113528506B (en) | DNA inversion system and application thereof and target DNA inversion method | |
| CN108265068A (en) | Recombinate arginine deiminase and its industrialization preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20120704 |