CN104745634A - Method for improving efficiency of expressing foreign protein by using insect baculovirus expression system - Google Patents
Method for improving efficiency of expressing foreign protein by using insect baculovirus expression system Download PDFInfo
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Abstract
The invention discloses a method for improving the efficiency of expressing a foreign protein by using an insect baculovirus expression system. The method comprises the following steps of culturing a stroma cell in a culture medium; performing heat shock on the stroma cell in the culture process of the stroma cell; inoculating a baculovirus; harvesting cell suspension and extracting the expressed foreign protein. By using the method disclosed by the invention, the insect baculovirus expression system can efficiently express the foreign protein; particularly, compared with the prior art, the method disclosed by the invention has the advantages that the expression quantity of the foreign protein can be improved by over 7.6 times, and the application of the expression system for industrially producing vaccine and other biological products on a large scale can be greatly widened.
Description
Technical field
The present invention relates to a kind of method utilizing insect baculovirus expression system to express foreign protein, particularly a kind of raising utilizes insect baculovirus expression system to express the method for foreign protein, belongs to exogenous protein expression preparing technical field.
Background technology
Over nearly 20 years, insect cell-baculovirus expression system (BEVS) is successfully applied to and have expressed a lot of foreign protein.Three good clones that compare are had to have good character at present, be applicable to research and the production of AcMNPB and BmNPV expression of recombinant proteins, it comprises: derive from IPLB-SF21AE (Sf21) clone of autumn mythimna separata ovary tissue and derivative strain Sf9 thereof, from BTI TN5B1-4 (High Five) clone of wild cabbage chi pot, and derive from BmN and the subbreed thereof of silkworm ovary.Wherein Sf9 is wherein Application comparison cell strain widely, and have now a lot of product all to apply baculovirus expression foreign protein, especially VLP is as vaccine.
Insect cell belongs to half attached cell, the foetal calf serum (FBS) containing 10% can be used to cultivate, also serum-free adherent culture can be carried out, in order to more adapt to scale operation, generally all carry out serum free suspension cultivation now, the existing BEVS of utilization system expression albumen is prepared vaccine and is all adopted the mode of suspension culture to carry out.Therefore, in whole technological process, the doubling time of clone, cell density, the ability of cell to viral susceptibility and cell expression exogenous gene become main Consideration.
Utilize baculovirus expression system successfully to develop the multiple vaccine product that gone on the market at present both at home and abroad, comprised people and use listing vaccine, listing vaccine for animals etc.By target gene through optimizing, then molecular biology method is passed through, build and obtain sufficient baculovirus, again by baculovirus infection insect cell, through obtaining target protein, obtaining satisfactory target product through various means of purification again, is the basic ideas utilizing insect-bacvdovirus system vaccine development product at present.The on the one hand protein produced of insect cell comparatively protokaryon and the easier glycosylation of eukaryotic expression system such as low, be protein more close to natural radioactivity state, comparatively 293 cells and expressing cho cell system have more advantage to utilize on the other hand baculovirus expression viral protein.Because above advantage, the multiple vaccine utilizing this expression system to produce is had to be in clinical phase or research and development phase.
At present, insect baculovirus expression system is used to carry out protein expression, although achieved the industrial scale of 1000L, but influence factor---" cell density effect " that in Baculovirus expression system use procedure, existence one is important, namely, when inoculating baculovirus, cell density should lower than 3 × 10
6cells/ml, more efficiently is should lower than 2 × 10
6cell/ml, can not improve higher than this cell density virus inoculation exogenous protein expression amount and even can decline.Although report, use perfusion culture technique, can improve cell density during virus inoculation, exogenous protein expression amount also can improve, and insect cell oxygen-consumption is high, uses the existing bio-reactor of perfusion culture to be difficult to maintain.And this systematic protein expression amount, only at about 15mg/L, brings larger challenge to the purifying in product later stage at present.Meanwhile, batch cultivation maximum cell density of insect cell in general commercialization serum free medium can reach 1 × 10
7cells/ml, in the serum free medium that some is special, batch cultivation can reach 2.3 × 10
7cells/ml, but the only virus inoculation when low density, can cause the serious waste of substratum.
Summary of the invention
Main purpose of the present invention is to provide a kind of raising to utilize insect baculovirus expression system to express the method for foreign protein efficiency, to overcome deficiency of the prior art.
For realizing aforementioned invention object, the technical solution used in the present invention comprises:
Raising utilizes insect baculovirus expression system to express a method for foreign protein efficiency, comprising:
(1) culture medium cell in the medium;
(2) in the culturing process of stroma cell, thermal shock is carried out to stroma cell;
(3) baculovirus is inoculated;
(4) foreign protein of harvested cell suspension and extraction expression.
Among a comparatively preferred embodiment, step (1) comprising: cultivate using Sf9 cell as stroma cell.
Further, the culture condition of step (1) mesostroma cell comprises: temperature is 25 ~ 28 DEG C, and mixing speed is 20 ~ 60rpm, and dissolved oxygen is 30 ~ 60%, and pH value is 6.0 ~ 6.4.
Among a comparatively preferred embodiment, the method comprises: inoculating stroma cell to cell density is in the medium 0.5 × 10
6~ 1.5 × 10
6cultivate after individual/ml, and before inoculation baculovirus, the stroma cell total amount in substratum reaches 6 × 10
6~ 1 × 10
7individual/ml.
Among a comparatively preferred embodiment, step (2) comprising: 60 ~ 72h after inoculation stroma cell, culture temperature is risen 1 ~ 5 DEG C, and maintains 1 ~ 8 hour, afterwards by temperature return to normal temperature for cultivation.
Further, described substratum is preferably insect cell serum free medium.
Among a comparatively preferred embodiment, step (3) comprising: inoculate baculovirus according to MOI=5 ~ 15.
Among a comparatively preferred embodiment, described baculovirus is preferably the recombinant baculovirus containing PCV-2 virus coat protein gene.
Among a comparatively preferred embodiment, the method comprises: adopt fed-batch process culture medium cell.
Among a comparatively preferred embodiment, the method also comprises: after inoculation baculovirus, intermittent injecting nutritive substance, described nutritive substance comprises the combination of any one or more in glucose, glutamine, peptone, yeast extract.
Relative to existing Baculovirus expression system, serum free medium in the inventive method, is utilized to cultivate insect cell, such as Sf9 insect cell, and not because " cell density effect " and at Growth of Cells to 1.5 ~ 3 × 10
6individual/ml time inoculation baculovirus promoter exogenous gene expression.The essence of " cell density effect " is caused to be after cell reaches certain cell density, the ratio that S in this period phase cell accounts for and total amount maximum, when cell density continues to raise, S phase cell proportion and quantity all decline very fast, even if so cell density when improving virus inoculation corresponding increase virus inoculation amount can not improve or even reduce the expression amount of foreign gene.When cell enlargement is to the density needed, cell is carried out to the thermal shock of certain hour, recover normal temperature for cultivation afterwards, in cell, S phase cell proportion significantly increases.S phase cell increases to virus infection and provides good condition.Virus infecting under high-cell density can be realized.Therefore adopt high-cell density virus inoculation in the present invention, such as, 0.6 ~ 1 × 10
7individual/ml carries out connecing poison, and inoculating cell density improve 3 ~ 6 times more originally.Improve S phase cell proportion in cell by thermal shock effect simultaneously, make unicellular output increased, increase under dual function in raising inoculating cell density and unicellular output, the expression amount of foreign protein after virus inoculation can be significantly improved.
Postscript, in traditional technology, after virus inoculation, the general mode adopting batch culture, does not carry out the operation of any extra-nutrition material.But in the present invention, carry out Fed batch fementation after virus inoculation, carry out nutritive substance supplement it, when ensureing to express foreign protein, cell desired nutritional material is sufficient.
In a word, compared with prior art, by in method of the present invention, cell density when can significantly improve virus inoculation, and then significantly improve the expression amount of foreign protein in unit volume, particularly, than existing virus inoculation technique, exogenous protein expression amount can improve more than 7.6 times, greatly can expand the application of this expression system in the biological products such as large-scale industrial production vaccine.
Accompanying drawing explanation
Fig. 1 receives for utilizing thermal shock to improve bio-reactor in one embodiment of the invention the process flow sheet that insect baculovirus expression system expresses foreign protein;
Fig. 2 be use thermal shock (embodiment 1) and do not use thermal shock (reference examples 1) condition under, the growth curve chart of cell in bio-reactor;
Fig. 3 is the examination criteria curve of a kind of kit standard product among one embodiment of this invention.
Embodiment
Low for expressing quantity existing when utilizing insect baculovirus expression system to carry out protein expression in prior art, substratum waste is serious waits deficiency, inventor is in studying for a long period of time and putting into practice in a large number, find very unexpectedly, employing thermal shock method can the amazing expression efficiency significantly improving insect baculovirus expression system.Find based on this, inventor is proposed technical scheme of the present invention, carries out specific explanations explanation as follows.
Among an embodiment of the present invention, provide a kind of raising and utilize insect baculovirus expression system to express the method for foreign protein efficiency, it comprises the following steps:
1) stirring type bioreactor culture medium cell is applied;
2) in culturing process, thermal shock is carried out to cell;
3) recombinant baculovirus is inoculated;
4) harvested cell suspension, testing goal protein content.
Preferably, step 1) in stroma cell used be Sf9 cell.
Preferably, step 3) in the baculovirus that adopts be recombinant baculovirus containing exogenous protein expression gene.
The bio-reactor that the present invention adopts with oblique three leaf oars or can be called elephant ear oar; Or bio-reactor used is disposable stirring type bioreactor.
Preferably, step 1) described in bioreactor culture cell technique comprise amplification technique, single-stage cultivate in seed cell used be flask suspension culture cell, amplification culture seed cell used is cultivated by bioreactor system and is obtained cell.Single-stage is cultivated as 14L bio-reactor single-stage training mode, and amplification culture is 14L to 42L amplification mode.Concrete amplification method is by cultured cells in shaking flask, transfers in bio-reactor and cultivate after dilution, continues dilution after reaching certain density, and import in the bio-reactor of new more volume and continue to cultivate, method is put into required scale successively.
Preferably, step 1) described in bioreactor culture stroma cell mode be fed-batch process.
Preferably, step 1) described in bioreactor culture cell condition be: temperature 25 ~ 28 DEG C, mixing speed is 20 ~ 60rpm, and dissolved oxygen (DO) is 30 ~ 60%, and pH value is 6.0 ~ 6.4.
Preferably, step 1) described in bioreactor culture cell substratum used be insect cell serum free medium (such as, doubly entering SF20112-4).
In a concrete embodiment, in the present invention, inoculating cell density is 0.5 ~ 1.5 × 10
6individual/ml, the cell total amount before inoculation reaches 0.6 ~ 0.9 × 10
7cells/ml.
Bioreactor culture pattern of the present invention is fed-batch process.
In the process of bioreactor culture stroma cell, thermal shock is carried out to cell, refers to: 60 ~ 72 hours after inoculation, culture temperature is risen 1 ~ 5 DEG C, and maintains 1 ~ 8 hour, afterwards that temperature return is normal.
Bio-reactor is expressed foreign protein and is inoculated recombinant baculovirus according to suitable proportion, and the ratio of virus inoculation is generally MOI=5 ~ 15.Total the english abbreviation of MOI and virus infection attached (Multiplicity Of Infection), be exactly the ratio of intuitively total virus number and cell count, determines virus inoculation amount by cell concn before sampling counting virus inoculation.
In the present invention, further preferred technical scheme is, step 3) described in virus culture process be feed-batch culture technique, namely, after virus inoculation, intermittent injecting nutritive substance, as arbitrary in glucose, glutamine, peptone, yeast extract etc. or its combination.
Wherein, the results of cell suspension can adopt harvesting approach in batches.
Preferably, step 4) detect in foreign protein and the cell suspension of results is carried out ultrasonication, suspended substance is carried out centrifugal, remove fragment, get supernatant, such as, PCV2-Cap protein quantification detection kit (article No.: P1001) can be used to detect the concentration (detection curve can consult Fig. 3) of object product in harvest liquid.
Below with reference to exemplary embodiments, more clear, complete description is carried out to technical scheme of the present invention.But embodiment of the present invention are not limited thereto.
Embodiment 1: the reactor that the present embodiment adopts is Sai Duolisi stirring-type 14L reactor and 42L reactor, for oblique three leaf oars, the stroma cell adopted is Sf9 cell, substratum is SF-SFM (Wo Mei Bioisystech Co., Ltd of Suzhou City), and the recombinant baculovirus containing foreign gene adopted is the recombinant baculovirus (Biotechnology Co., Ltd. on the Hai Midi) with PCV-2 glutelin.Its technical process comprises:
(1) preparation of shaking flask and bio-reactor
Shaking flask thoroughly cleaned, masking foil wraps up shaking flask respectively, carries out autoclaving, 121 DEG C, and 30min, naturally cools to room temperature, is transferred to dry for standby in baking oven.
Thoroughly cleaned by bio-reactor tank body, carried out by pH after demarcation and DO electrode is installed on tank body, wrapping pipe joint also carries out testing Lous experiment.Complete artifact reactor and carry out high pressure steam sterilization, sterilising conditions is 121 DEG C, 30min.Be connected on reactor by aseptic for cell growth medium that sterile filtration is good, tank body is imported by peristaltic pump, the minimum add-on of substratum with submergence electrode probe for standard, and open balance and the preheating that temperature of reactor, stirring and ventilation control to carry out substratum, and DO electrode is corrected, spend the night stand-by.
(2) stroma cell prepared by bio-reactor
Concrete steps are, cultured cells in shaking flask are imported in bio-reactor, and supplement fresh culture, make the density of cell suspension in bio-reactor be 1 × 10
6individual/ml, setting bioreactor culture processing condition are: temperature is 27 DEG C, and mixing speed is 60rpm, DO 40%, pH 6.3.Sample counting cells density every day, when cell density reaches 3 × 10
6individual/ml, transfers to culture in 42L reactor, and supplemental medium, make cell density be 1 × 10
6individual/ml, setting culture condition is 27 DEG C, and mixing speed is 35rpm, DO 40%, pH 6.3.Cell growth cycle is generally 72 hours.
(3) thermal shock is carried out
After cell sampling, counting cells density.After cultivation 72h, cell density reaches 8 × 10
6cells/ml, raises culture temperature to 29.5 DEG C, and maintains 8 hours, renewal cultivation temperature to 27 DEG C afterwards.
(4) virus inoculation
According to the recombinant baculovirus of MOI=1 inoculation containing foreign gene.
(5) continue to cultivate 72h, harvested cell suspension.
(6) cell suspension of results is carried out three multigelations, using ELISA to detect foreign protein concentration is 494mg/L.
Reference examples 1: conversion unit and the raw material of the employing of this reference examples are identical with embodiment, and its technical process comprises:
1) preparation of shaking flask and bio-reactor
Shaking flask thoroughly cleaned, masking foil wraps up shaking flask respectively, carries out autoclaving, 121 DEG C, and 30min, naturally cools to room temperature, is transferred to dry for standby in baking oven.
Thoroughly cleaned by bio-reactor tank body, carried out by pH electrode after demarcation and DO electrode is installed on tank body, wrapping pipe joint also carries out testing Lous experiment.Complete artifact reactor and carry out high pressure steam sterilization, sterilising conditions is 121 DEG C, 30min.Be connected on reactor by aseptic for cell growth medium that sterile filtration is good, tank body is imported by peristaltic pump, the minimum add-on of substratum with submergence electrode probe for standard, and open balance and the preheating that temperature of reactor, stirring and ventilation control to carry out substratum, DO is corrected, spends the night stand-by.
2) stroma cell prepared by bio-reactor
Concrete steps are, cultured cells in shaking flask are imported in bio-reactor, and supplement fresh culture, make the density of cell suspension in bio-reactor be 1 × 10
6individual/ml, setting bioreactor culture processing condition are: temperature is 27 DEG C, and mixing speed is 60rpm, DO 40%, pH 6.3.Sample counting cells density every day, when cell density reaches 3 × 10
6individual/ml, transfers to culture in 42L reactor, and supplemental medium, make cell density be 0.8 × 10
6individual/ml, setting culture condition is 27 DEG C, and mixing speed is 35rpm, DO 40%, pH 6.3.Cell growth cycle is generally 12 hours.
3) virus inoculation
After cell sampling, counting cells density.After cultivation 12h, cell density reaches 1.5 × 10
6cells/ml, according to the recombinant baculovirus of MOI=1 inoculation containing foreign gene.
5) continue to cultivate 72h, harvested cell suspension.
6) cell suspension of results is carried out three multigelations, using ELISA to detect foreign protein concentration is 65mg/L.
Embodiment 2
The reactor that the present embodiment adopts is Sai Duolisi stirring-type 14L reactor and 42L reactor, for oblique three leaf oars, the stroma cell adopted is Sf9 cell, substratum is SF-SFM (Wo Mei Bioisystech Co., Ltd of Suzhou City), and the recombinant baculovirus containing foreign gene adopted is the recombinant baculovirus (Biotechnology Co., Ltd. on the Hai Midi) containing GFP foreign gene.Its technical process comprises:
(1) preparation of shaking flask and bio-reactor
Shaking flask thoroughly cleaned, masking foil wraps up shaking flask respectively, carries out autoclaving, 121 DEG C, and 30min, naturally cools to room temperature, is transferred to dry for standby in baking oven.
Thoroughly cleaned by bio-reactor tank body, be installed on tank body after being demarcated by pH and DO electrode, wrapping pipe joint also carries out testing Lou experiment.Complete artifact reactor and carry out high pressure steam sterilization, sterilising conditions is 121 DEG C, 30min.Be connected on reactor by aseptic for cell growth medium that sterile filtration is good, tank body is imported by peristaltic pump, the minimum add-on of substratum for standard with submergence electrode probe, and is opened temperature of reactor, stirring and ventilation and is controlled to carry out balance and the preheating of substratum, spends the night stand-by.
(2) stroma cell prepared by bio-reactor
Concrete steps are, cultured cells in shaking flask are imported in bio-reactor, and supplement fresh culture, make the density of cell suspension in bio-reactor be 1 × 10
6individual/ml, setting bioreactor culture processing condition are: temperature is 27 DEG C, and mixing speed is 60rpm, DO 40%, pH 6.3.Sample counting cells density every day, when cell density reaches 3 × 10
6individual/ml, transfers to culture in 42L reactor, and supplemental medium, make cell density be 1 × 10
6individual/ml, setting culture condition is 27 DEG C, and mixing speed is 35rpm, DO 40%, pH 6.3.Cell growth cycle is generally 72 hours.
(3) thermal shock is carried out
After cell sampling, counting cells density.After cultivation 72h, cell density reaches 8 × 10
6cells/ml, raises culture temperature to 29.5 DEG C, and maintains 8 hours, renewal cultivation temperature to 27 DEG C afterwards.
(4) virus inoculation
According to the recombinant baculovirus of MOI=1 inoculation containing foreign gene.
(5) continue to cultivate 72h, harvested cell suspension, living cell counting density is 7.3 × 10
6individual/ml, 3000rpm, centrifugal 5min.
(6) use cold PBS gravity treatment cell precipitation, 3000rpm, centrifugal 5min, repeat 3 times, cell density is diluted to 1 × 10
6individual/ml prepares cell suspension.
Reference examples 2:
Conversion unit and the raw material of the employing of this reference examples are identical with embodiment, and its technical process comprises:
1) preparation of shaking flask and bio-reactor
Shaking flask thoroughly cleaned, masking foil wraps up shaking flask respectively, carries out autoclaving, 121 DEG C, and 30min, naturally cools to room temperature, is transferred to dry for standby in baking oven.
Thoroughly cleaned by bio-reactor tank body, be installed on tank body after being demarcated by pH and DO electrode, wrapping pipe joint also carries out testing Lou experiment.Complete artifact reactor and carry out high pressure steam sterilization, sterilising conditions is 121 DEG C, 30min.Be connected on reactor by aseptic for cell growth medium that sterile filtration is good, tank body is imported by peristaltic pump, the minimum add-on of substratum for standard with submergence electrode probe, and is opened temperature of reactor, stirring and ventilation and is controlled to carry out balance and the preheating of substratum, spends the night stand-by.
2) stroma cell prepared by bio-reactor
Concrete steps are, cultured cells in shaking flask are imported in bio-reactor, and supplement fresh culture, make the density of cell suspension in bio-reactor be 1 × 10
6individual/ml, setting bioreactor culture processing condition are: temperature is 27 DEG C, and mixing speed is 60rpm, DO 40%, pH 6.3.Sample counting cells density every day, when cell density reaches 3 × 10
6individual/ml, transfers to culture in 42L reactor, and supplemental medium, make cell density be 0.8 × 10
6individual/ml, setting culture condition is 27 DEG C, and mixing speed is 35rpm, DO 40%, pH 6.3.Cell growth cycle is generally 12 hours.
3) virus inoculation
After cell sampling, counting cells density.After cultivation 12h, cell density reaches 1.5 × 10
6cells/ml, according to the recombinant baculovirus of MOI=1 inoculation containing GFP gene.
5) continue to cultivate 72h, harvested cell suspension, living cell counting density is 1.2 × 10
6individual/ml
6) use cold PBS gravity treatment cell precipitation, 3000rpm, centrifugal 5min, repeat 3 times, cell density is diluted to 1 × 10
6individual/ml prepares cell suspension.
Detect the fluorescin intensity in embodiment 2 and reference examples 2: in black 96 orifice plate, add sample in the following order:
Sample 1: Sf9 (baculovirus of inoculation not containing the foreign gene) cell suspension using cold PBS to prepare, cell density is 1 × 10
6individual/ml
Sample 2: with Sf9 (baculovirus of inoculation containing GFP gene) cell suspension prepared by PBS in embodiment 2, cell density is 1 × 10
6individual/ml.
Sample 3: with Sf9 (baculovirus of inoculation containing GFP gene) cell suspension prepared by PBS in reference examples 2, cell density is 1 × 10
6individual/ml.
Use Biotek H1 fluorescence microplate reader, setting exciting light 485nm, utilizing emitted light 535nm carries out fluoroscopic examination.Detected result is as shown in the table:
| Sample 1 | Sample 2 | Sample 3 | PBS | |
| A | 16 | 38654 | 59328 | 15 |
| B | 19 | 39700 | 62723 | 16 |
| C | 15 | 40103 | 60209 | 15 |
| D | 17 | 40059 | 63800 | 13 |
| E | 17 | 41216 | 63360 | 19 |
| F | 15 | 41747 | 64043 | 16 |
| G | 17 | 41989 | 63255 | 17 |
| H | 12 | 41520 | 63540 | 21 |
| Mean value | 14.33333 | 36110 | 55584.56 | 15.11111 |
As can be seen from the above results, in embodiment 2, fluorescent value is (55584.56-15.11111) × 7.3=405657, fluorescent value (36110-15.11111) × 1.2=43314 in reference examples 2 is so 9.3 times of fluorescent value in reference examples 2 in embodiment 2.
In addition, inventor goes back the method with reference to previous embodiment and reference examples, utilize insect baculovirus expression system, using Sf9 cell as stroma cell, inoculate the recombinant baculovirus with pig parvoviral coat protein gene respectively, with the recombinant baculovirus of Pestivirus suis coat protein gene, carry out the expression of foreign protein, show after tested, adopt method of the present invention, the expression efficiency of foreign protein on average can be made to improve on 7.6 times.
Technology contents of the present invention and technical characteristic have disclosed as above; but those of ordinary skill in the art still may do all replacement and the modification that do not deviate from spirit of the present invention based on teaching of the present invention and announcement; be to be understood that; scope should be not limited to the content that embodiment discloses; and various do not deviate from replacement of the present invention and modification should be comprised, and contained by present patent application claim.
Claims (10)
1. raising utilizes insect baculovirus expression system to express a method for foreign protein efficiency, it is characterized in that comprising:
(1) culture medium cell in the medium;
(2) in the culturing process of stroma cell, thermal shock is carried out to stroma cell;
(3) baculovirus is inoculated;
(4) foreign protein of harvested cell suspension and extraction expression.
2. raising according to claim 1 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that step (1) comprising: cultivate using Sf9 cell as stroma cell.
3. raising according to claim 1 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that, the culture condition of step (1) mesostroma cell comprises: temperature is 25 ~ 28 DEG C, mixing speed is 20 ~ 60rpm, dissolved oxygen is 30 ~ 60%, and pH value is 6.0 ~ 6.4.
4. the raising according to any one of claim 1-3 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that comprising: inoculating stroma cell to cell density is in the medium 0.5 × 10
6~ 1.5 × 10
6cultivate after individual/ml, and before inoculation baculovirus, the stroma cell total amount in substratum reaches 6 × 10
6~ 1 × 10
7individual/ml.
5. the raising according to any one of claim 1-3 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that, step (2) comprising: 60 ~ 72h after inoculation stroma cell, culture temperature is risen 1 ~ 5 DEG C, and maintain 1 ~ 8 hour, afterwards by temperature return to normal temperature for cultivation.
6. the raising according to any one of claim 1-3 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that described substratum is insect cell serum free medium.
7. raising according to claim 1 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that comprising: inoculate baculovirus according to MOI=5 ~ 15.
8. the raising according to claim 1 or 7 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that described baculovirus is the recombinant baculovirus comprising foreign protein genes.
9. the raising according to any one of claim 1-3,7 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that comprising: adopt fed-batch process culture medium cell.
10. the raising according to any one of claim 1-3,7 utilizes insect baculovirus expression system to express the method for foreign protein efficiency, it is characterized in that comprising: after inoculation baculovirus, intermittent injecting nutritive substance, described nutritive substance comprises the combination of any one or more in glucose, glutamine, peptone, yeast extract.
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| CN112041450A (en) * | 2018-04-12 | 2020-12-04 | 生命技术公司 | Chemically defined baculovirus expression systems and cell culture media |
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