CN109468268A - A kind of method and application for cultivating HEK-293T cell strain efficient secretory expression swine fever E2 albumen - Google Patents
A kind of method and application for cultivating HEK-293T cell strain efficient secretory expression swine fever E2 albumen Download PDFInfo
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Abstract
The invention discloses a kind of methods for cultivating HEK-293T cell strain efficient secretory expression swine fever E2 albumen, this method is that serum-containing media is added in bioreactor, HEK-293T cell is inoculated, detects concentration of glucose by culture medium elder generation circulated at low velocity, again after high-speed circulating；Every 22-26h detection cell consumption sugar, when residual sugar is lower than 2g/L, addition glucose to 3.8~4.2g/L；When daily sugar consumption is more than 1.5g/L, culture medium is removed, serum free medium is added；Sugar amount is consumed according to cell, adds glucose；Every 4-5 days collection part volumes, and refill new serum free medium.For the present invention by optimization parameters, primary experiment can at most collect 15 batches expression albumen, and production capacity is high.And condition of culture is stablized, repeatability is high.The method of the present invention makes the cell density of culture high, and expression quantity is high.Subunit vaccine low manufacture cost, cost performance is high, and application prospect is big.
Description
Technical field
The present invention relates to a kind of methods and application for cultivating HEK-293T cell strain efficient secretory expression swine fever E2 albumen.
Background technique
Swine fever (Classical swine fever, CSF) is commonly called as " rinderpest ", is by swine fever virus (Classical
Swine fever virus, CSFV) caused by one kind it is acute, fever, contagious disease, have it is highly infectious and lethal
Property, it is to seriously threaten one of Infectious Diseases of pig breeding industry, the Ministry of Agriculture, China is defined as a kind of zoonosis.Swine fever exists
China is still popular, according to statistics, China because disease death pig in there is 30% or more to be swine fever caused by, and there is no be directed at present
The specific drug of swine fever, therefore, specification inoculation hog cholera vaccine is the effective ways that swine fever morbidity and mortality are effectively reduced.
CSFV belongs to flaviviridae (Flaviridae) pestivirus (Pestivirus), is single strand plus RNA virus.Base
Because group is about 12.3kb, single open reading frame (ORF) is contained only, encodes 4 kinds of structural proteins (c, E0, E1, E2) and 8 kinds of non-knots
Structure albumen (Npro, P7, NS2, NS3, NS4A, NS4B, NS5A, NS5B).Wherein, envelope glycoprotein E2 is the main protection of swine fever
Property antigen, CSFV infection when can induce body generate neutralizing antibody, be mono- most effective immunogene of CSFV, thus become grind
Send out the first choice of CSFV novel subunit vaccine.After traditional hog cholera vaccine immune swine, it can generate be directed to E2 albumen and E0 in vivo
The specific antibody of albumen；The antibody that anti-swine fever E2 albumen can be only generated after swine fever E2 subunit vaccine is immune, can be used as label
Vaccine distinguishes vaccine immunity pig and wild virus strain infection by detection E0 albumen, to reach the purification on pig farm.
The present inventor constructs a kind of efficient stable people secretion in the application for a patent for invention application No. is 201510887848.5
Property expression CSFV E 2 protein recombinant cell lines, expression quantity of the cell line in Tissue Culture Flask can reach 0.2g/
L.In order to further increase the expression of cell, the manufacturing cost of vaccine is reduced, and further realize the rule of production of vaccine
Modelling, on the basis of original, the pilot scale culture for studying the cell using bioreactor and serum free medium is raw for we
Production method.But existing bioreactor expression has the disadvantage in that expression quantity is lower to cell expression virus E2 albumen, epidemic disease
Seedling higher cost；Express small scale, it is impossible to be used in industrially scalable metaplasia produces；Cell culture condition can not be controlled during expression
System, not can be carried out process optimization；There is cell to grow contact inhibition phenomenon in cell in culture dish, cell density is low, growth cycle
It is short；Culture dish working condition is unstable, and difference is big between batch.
Summary of the invention
The purpose of the present invention is to provide a kind of sides for cultivating HEK-293T cell strain efficient secretory expression swine fever E2 albumen
Method.
The technical solution used in the present invention is:
A method of culture HEK-293T cell strain efficient secretory expression swine fever E2 albumen, comprising the following steps:
1) pretreatment of bioreactor；
2) the DMEM complete medium containing serum is added toward pretreated bioreactor, does steriling test overnight,
After the completion of steriling test；Vitellophag is seeded in infusion bag with the DMEM complete medium containing serum again, and is connect thereto
Kind HEK-293T cell, staticaccelerator adsorption；Culture medium elder generation low speed 130-170ml/min is recycled into 25-35min, again high speed 180-
220 ml/min recycle sample detection concentration of glucose after 25-35min, as the sugared concentration of starting；
Cell culture temperature is set: 36-37.5 DEG C, cell culture pH:7.0-7.2, cell culture DO:30%-80%, being turned
Speed: 72-77rpm, circulation rate: 200-300ml/min, air mass flow: 180-220cc/min；
3) culture solution detection cell consumption sugar was taken to add concentration of glucose when residual sugar is lower than 2g/L every 22-26 hours
To 3.8~4.2g/L；
4) when daily sugar consumption is more than 1.5g/L, the culture medium containing serum is removed, after PBS cleaning reactor is added, is added
Enter serum free medium to be expressed；Cell culture temperature is set simultaneously: 35-36.5 DEG C, cell culture pH:7.2-7.4, cell
Cultivate DO:30%-80%, air mass flow: 200cc/min, revolving speed: 58-62rpm, circulation rate: 200-300ml/min；
5) every 22~26 hours taking-up culture solution detection cell consumption sugar, and concentration of glucose is added to 3.8~4.2g/
L；Sugar amount is consumed according to the cell of last time detection simultaneously, divides in next 22~26 hours 11~13 times and mends the consumption sugar amount
It returns；Such circulate operation；
6) culture medium of 2/3-4/5 volume was collected every 4-5 days, and refills new serum free medium.
Preferably, the pretreatment of the bioreactor includes checking that whether there is or not breakage, torrent bags, infusion bag for consumptive material
Inflation leak detection is overnight；DO electrode energization polarization 6h or more；After calibration electrodes sterilizing in second day, tinning connecting line, system connection
Air-tightness is checked after good, PBS buffer solution is added, and by the scraps of paper carrier soaked overnight in reactor, third day removes PBS.
Preferably, the bioreactor is 4L reactor.
Preferably, in step 2), in the DMEM complete medium containing serum contain 9.8~10.2% fetal calf serums, 4.4~
The glucose of 4.6g/L.
Preferably, in step 2), total dosage of the DMEM complete medium containing serum is 3.8~4.2L；HEK-293T is thin
Inoculum concentration 1-2 × 10 of born of the same parents9It is a.
Preferably, in step 2), the time of staticaccelerator adsorption is 0.8~1.2h.
Preferably, in step 2) and step 4), with 7~8%NaHCO3And CO2PH is adjusted, N is used2Adjust dissolved oxygen DO.
Preferably, it is 4.15~4.25g/L that sugared concentration is originated in step 2).
Preferably, in step 3) and step 5), concentration of glucose is added to 3.8 with the glucose solution of 280-320g/L
~4.2g/L.
Preferably, in step 4), the formula of serum free medium is the CD 293TGE Medium containing 4nM caffeine.
Wherein, CD 293TGE Medium is ACROBiosystems product, and coffee is because of sigma Products
Preferably, in step 6), the expression quantity of albumen in the culture medium that detection every batch of is collected, when expression of cellular proteins amount is low
In 0.40g/L or consumption sugar amount lower than after 1.2g/L, stop culture.
The beneficial effects of the present invention are:
(1) for the present invention by optimization parameters, primary experiment can at most collect 15 batches expression albumen, and production capacity is high.
And condition of culture is stablized, repeatability is high.
(2) the method for the present invention makes the cell density of culture high, and expression quantity is high.Subunit vaccine low manufacture cost, cost performance
Height, application prospect are big.
(3) liquid feeding, receipts liquid are all controlled by system during the method for the present invention, are reduced operating procedure, are reduced contamination probability.
Detailed description of the invention
Fig. 1 is the electrophoresis detection figure of cell express express target protein (swine fever E2) amount in each batch of culture solution collected, 1- in figure
9: respectively expressing the 1st, 3,5,7,9,11,13,14,15 batch of albumen.
Fig. 2 is the electrophoresis detection figure of cell express express target protein (swine fever E2) amount in each group culture solution, 1-3: culture dish table
The albumen 1-3 reached batches；4-6: 1-3 batches of albumen of bioreactor expression of the present invention.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below.
The culture production method of the HEK-293T cell strain of 1 efficient secretory expression swine fever E2 albumen of embodiment
One, the amplification of seed cell
The HEK-293T cell strain that the expression swine fever E2 albumen saved is taken out from nitrogen storage tank, is placed in 37 DEG C of constant temperature
Melt rapidly in water-bath, 800rpm is centrifuged 5min and removes cells frozen storing liquid, is cultivated completely with the DMEM containing 10% fetal calf serum
Outstanding be placed in 150mM Tissue Culture Dish of base weight is cultivated in carbon dioxide incubator.Every 2-3 days, when cell density reaches
It is expanded in 150mM Tissue Culture Dish when 90% or more by 1:5, until cell number reaches 109Biological respinse is transferred to when above
In device.
Two, the culture process method of bioreactor
1) checking consumptive material, whether there is or not breakages, record batch.Torrent bag, infusion bag inflation leak detection (notice that flow of aerating air is other overnight
Excessively big, filter membrane can be crushed causes sack to be destroyed).DO electrode energization polarization 6h or more.After calibration electrodes sterilizing in second day, dress
Tank connecting line, system will check air-tightness after connecting, squeeze into PBS buffer solution 2.5L, and the scraps of paper carrier in reactor is soaked
Overnight, PBS is got in third day to bubble.
2) it is complete that high sugar (concentration of glucose 4.5g/L) DMEM of 2.5L containing 10% fetal calf serum is added into above-mentioned reactor
Full culture medium, 37 DEG C are done steriling test overnight.After the completion of steriling test, vitellophag is inoculated with 1.5L DMEM complete medium
To infusion bag, inoculum concentration 1-2 × 109A cell, staticaccelerator adsorption 1h, low speed 150ml/min recycle 30min, high speed 200ml/
Sample detection sugar concentration is 4.2g/L after min recycles 30min, as the sugared concentration of starting.Setting cell culture temperature: 37 DEG C ±
0.5 DEG C, cell culture pH:7.0-7.2, cell culture DO:30%-80%, revolving speed: 75rpm, circulation rate: 200-300ml/
Min, air mass flow: 200cc/min.Use 7.5%NaHCO3And CO2PH is adjusted, N is used2Adjust dissolved oxygen.
3) glucose is detected every 24 hours taking-up culture solutions, detection cell consumption sugar assesses the growing state of cell.Grape
Sugar is measured using the Glucose estimation kit (glucose oxidase-peroxidase method) of Shanghai Rong Sheng biology Pharma Inc.:
It will indicate the equivalent reagent mixing 1mL of R1 and R2,20 μ L samples, 37 DEG C of water-bath 13min, after colour developing, in wavelength 505mM be added
Place reads light absorption value, and compares with standard items, measures glucose content in culture solution.Sugar consumption (g/L)=former culture medium grape for 24 hours
Glucose content (g/L) after sugared content (g/L)-is cultivated 24 hours.When residual sugar is lower than 2g/L, mended with the glucose of 300g/L
It fills, glucose is added to 4g/L.
4) when daily sugar consumption is more than 1.5g/L, the culture medium by original containing serum is got, and squeezes into 2L PBS cleaning reactor,
4L serum free medium (the CD 293TGE Medium containing 4nM caffeine) is squeezed into afterwards to be expressed.Simultaneously by reactor item
Part is set as cell culture temperature: 35~36..5 DEG C, cell culture pH:7.2-7.4, cell culture DO:30%-80%, air
Flow: 200cc/min, revolving speed: 60rpm, circulation rate: 200-300ml/min.Use 7.5%NaHCO3And CO2PH is adjusted, N is used2
Adjust dissolved oxygen.
5) glucose is detected every 24 hours taking-up culture solutions, detection cell consumption sugar is added to the glucose of 300g/L
4g/L.Sugar is consumed according to daily cell, every natural gift 12 times were automatically replenished every two hours systems.
6) it was changed every 4-5 days and collects 3L culture medium, and change to the new serum free medium of 3L.Detect the expression of every batch of albumen
Amount, when expression quantity is substantially reduced, (expression quantity is lower than 0.40g/L) or glucose consumption obviously tails off and (consumes sugar amount lower than 1.2g/
L after), tank under bioreactor takes upper middle lower part Multifunctional paper-separating sheet of paper, microscopically observation.Cell density is substantially uniform, and part cell is also
Active, dead cell is more.
This experiment in total collect 15 batches, with the applicant's patent (number of patent application: CN201710728873.8, one plant
Express CSFV E 2 protein monoclonal antibody hybridoma cell line CSFV-3H3G6 and antibody and kit) in anti-swine fever E2
Monoclonal antibody as primary antibody, Western Blot detects the expression quantity of every batch of albumen.Wherein every batch of albumen can be higher than
0.2g/L, multiple batches of to be greater than 1g/L, wherein highest expression quantity is up to 1.5g/L.Process flow is shown in Table 1, cell expression quantity and daily
Sugar consumption is shown in Table 1 and Fig. 1.
The amount of cell express express target protein and sugar consumption in 1 biological reactor process process of table, each batch of culture solution
The expression of 2 bioreactor of embodiment and the experiment of culture dish expression
Culture dish expression:
1) according to the condition of culture of 1 bioreactor of embodiment, different cells is accessed in proportion in Tissue Culture Dish
Number, and with 300g/L glucose and 7.5%NaHCO3Reaction condition in culture dish is adjusted consistent with bioreactor.
2) after cell covers with, 20mL serum free medium is changed, and with 300g/L glucose and 7.5%NaHCO3It will training
The reaction condition adjusting supported in ware is consistent with bioreactor.15mL serum-free was changed every 4 days supports base, it is every with bioreactor
Batch compares expressing quantity.
As a result, being expressed with Tissue Culture Dish, 3 batches of expression samples can only be collected, and expression quantity can only achieve 0.2g/L or so
(as shown in Figure 2).
Influence of the different pH value of embodiment 3 to protein yield
Comparative example 1: in order to compare influence of the condition of different pH to protein expression, we utilize 4L bioreactor, setting
Different pH carries out culture expression, and specific experiment process is shown in embodiment 1 (in addition to setting the pH in 1 step 4) of embodiment to
7.0-7.1, other conditions with embodiment 1), concrete outcome is as shown in table 2.
The effect detection of HEK-293T cell strain expression swine fever E2 albumen under the different condition of culture of table 2
| Group | Expression condition | Express number of days | Highest protein yield (g/L) | Change the liquid time | Receive liquid batch |
| Comparative example 1 | 7.0-7.1 | 23 days | 0.62 | Every 3 days | 6 |
| Embodiment 1 | 7.2-7.4 | 62 days | 1.52 | Every 4 days | 15 |
From Table 2, it can be seen that by comparing, when slightly lower (7.0-7.1) is arranged in pH, cell culture medium hold time compared with
It is short, it must just change liquid within average 3 days, and cell growth state is poor, density is low, and expression quantity is low.It is capable of the number of days of express express target protein
Only 23 days, highest protein yield can only cross 0.62g/L, can only receive to obtain 6 wholesale zymotic fluids；And the method for the present invention (pH 7.2-7.4)
The number of days of cell high-efficient express express target protein can be made up to 62 days length, highest protein yield can harvest altogether up to 1.52g/L
15 wholesale zymotic fluids have been obviously improved the yield and timeliness of cell secretion destination protein.
Influence of the different cultivation temperatures of embodiment 4 to protein yield
Comparative example 2: the influence for more different cultivation temperatures to protein expression, we utilize 4L bioreactor, if
Fixed different pH carries out culture expression, and specific experiment process is shown in embodiment 1 (in addition to by the cell culture in 1 step 4) of embodiment
Temperature replaces with 30 DEG C ± 0.5 DEG C, and other conditions are consistent), concrete outcome is as shown in table 3.
The effect detection of HEK-293T cell strain expression swine fever E2 albumen under 3 condition of different temperatures of table
From table 3 it is observed that by comparing, when cultivation temperature is set as 30 DEG C ± 0.5 DEG C, cell culture basal growth is slow
Slowly, liquid is changed within average 6 days, and cell growth state is poor, density is low, and expression quantity is low.The number of days for capableing of express express target protein is 40 days,
Highest protein yield can only cross 0.6g/L, can only receive to obtain 7 wholesale zymotic fluids；And the method for the present invention (cultivation temperature is 35~36.5 DEG C)
The number of days of cell high-efficient express express target protein can be made up to 62 days length, highest protein yield can harvest altogether up to 1.52g/L
15 wholesale zymotic fluids have been obviously improved the yield and timeliness of cell secretion destination protein.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (10)
1. a kind of method for cultivating HEK-293T cell strain efficient secretory expression swine fever E2 albumen, which is characterized in that including following
Step:
1) pretreatment of bioreactor；
2) the DMEM complete medium containing serum is added toward pretreated bioreactor, does steriling test overnight, it is sterile
After having inspected；Vitellophag is seeded in infusion bag with the DMEM complete medium containing serum again, and is inoculated with thereto
HEK-293T cell, staticaccelerator adsorption；Culture medium elder generation low speed 130-170ml/min is recycled into 25-35min, again high speed 180-
220ml/min recycles sample detection concentration of glucose after 25-35min, as the sugared concentration of starting；
Setting cell culture temperature: 36-37.5 DEG C, cell culture pH:7.0-7.2, cell culture DO:30%-80%, revolving speed:
72-77rpm, circulation rate: 200-300ml/min, air mass flow: 180-220cc/min；
3) culture solution detection cell consumption sugar was taken to be added to concentration of glucose when residual sugar is lower than 2g/L every 22-26 hours
3.8~4.2g/L；
4) when daily sugar consumption is more than 1.5g/L, the culture medium containing serum is removed, after PBS cleaning reactor is added, nothing is added
Blood serum medium is expressed；Cell culture temperature is set simultaneously: 35-36.5 DEG C, cell culture pH:7.2-7.4, cell culture
DO:30%-80%, air mass flow: 200cc/min, revolving speed: 58-62rpm, circulation rate: 200-300ml/min；
5) every 22~26 hours taking-up culture solution detection cell consumption sugar, and concentration of glucose is added to 3.8~4.2g/L；Together
When sugar amount consumed according to the cell of last time detection, the consumption sugar amount is refilled in next 22~26 hours points for 11~13 times；Such as
This circulate operation；
6) culture medium of 2/3-4/5 volume was collected every 4-5 days, and refills new serum free medium.
2. being checked the method according to claim 1, wherein the pretreatment of the bioreactor includes
Whether there is or not breakages for consumptive material, and torrent bag, infusion bag inflation leak detection are overnight；DO electrode energization polarization 6h or more；Calibration electrodes are gone out within second day
After bacterium, tinning connecting line, system will check air-tightness after connecting, and PBS buffer solution be added, by the scraps of paper carrier in reactor
Soaked overnight, third day remove PBS.
3. the method according to claim 1, wherein the bioreactor is 4L reactor.
4. the method according to claim 1, wherein containing in the DMEM complete medium containing serum in step 2)
There are 9.8~10.2% fetal calf serums, the glucose of 4.4~4.6g/L.
5. the method according to claim 1, wherein in step 2), the DMEM complete medium containing serum it is total
Dosage is 3.8~4.2L；Inoculum concentration 1-2 × 10 of HEK-293T cell9It is a.
6. the method according to claim 1, wherein the time of staticaccelerator adsorption is 0.8~1.2h in step 2).
7. the method according to claim 1, wherein in step 2) and step 4), with 7~8%NaHCO3And CO2
PH is adjusted, N is used2Adjust dissolved oxygen DO.
8. the method according to claim 1, wherein in step 3) and step 5), with the grape of 280-320g/L
Concentration of glucose is added to 3.8~4.2g/L by sugar juice.
9. the method according to claim 1, wherein the formula of serum free medium is to contain 4nM in step 4)
The 293 TGE Medium of CD of caffeine.
10. the method according to claim 1, wherein detecting albumen in the culture medium that every batch of is collected in step 6)
Expression quantity, when expression of cellular proteins amount lower than 0.40g/L or consumption sugar amount be lower than 1.2g/L after, stop culture.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111718900A (en) * | 2020-06-19 | 2020-09-29 | 泉州台商投资区忆品茶业有限公司 | Amplification composition for in vitro stable amplification of high-purity and high-cytotoxic-activity NK cells |
| CN114409745A (en) * | 2021-06-04 | 2022-04-29 | 南方医科大学 | Production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102038944A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing swine fever live vaccine by using bioreactor |
| CN105331636A (en) * | 2015-12-04 | 2016-02-17 | 广州伯尼兹生物科技有限公司 | Recombination cell line for stable expression of classical swine fever virus E2 and application thereof |
| CN107974432A (en) * | 2017-08-11 | 2018-05-01 | 浙江海隆生物科技有限公司 | Culture method and application of CHO cell strain capable of efficiently secreting and expressing hog cholera E2 protein |
-
2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102038944A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing swine fever live vaccine by using bioreactor |
| CN105331636A (en) * | 2015-12-04 | 2016-02-17 | 广州伯尼兹生物科技有限公司 | Recombination cell line for stable expression of classical swine fever virus E2 and application thereof |
| CN107974432A (en) * | 2017-08-11 | 2018-05-01 | 浙江海隆生物科技有限公司 | Culture method and application of CHO cell strain capable of efficiently secreting and expressing hog cholera E2 protein |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111718900A (en) * | 2020-06-19 | 2020-09-29 | 泉州台商投资区忆品茶业有限公司 | Amplification composition for in vitro stable amplification of high-purity and high-cytotoxic-activity NK cells |
| CN114409745A (en) * | 2021-06-04 | 2022-04-29 | 南方医科大学 | Production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein |
| CN114409745B (en) * | 2021-06-04 | 2022-07-01 | 南方医科大学 | Production method for efficiently secreting and expressing porcine epidemic diarrhea virus S1 protein |
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