CN106381287A - Method for culturing animal cells for over expression of lactic dehydrogenase C - Google Patents
Method for culturing animal cells for over expression of lactic dehydrogenase C Download PDFInfo
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Abstract
The invention discloses a method for culturing CHO cells for over expression of lactic dehydrogenase C (LDH-C); an LDH-C enzymatic reaction substrate, such as sodium lactate, sodium pyruvate and the like, is directly added in a culture medium, a culture environment with a state of high lactic acid concentration is simulated, and the lactic acid production amount in LDH-C over expression recombinant cell culture process is ultimately achieved; the cell survival rate and the anti-apoptosis effect are improved.
Description
Technical field：
The invention belongs to biological technical field, more specifically disclose one kind and be used for overexpression Lactate dehydrogenase C（lactate dehydrogenase C）The cultural method of zooblast.
Background technology：
Between the past few decades, with Chinese hamster ovary cell（CHO）Zooblast for representing is widely used in expression (Jayapal, the KP. of recombiant protein
et al., (2007) Chem Eng Prog
103:40-43).The expression of recombiant protein depends on integration viable cell density and the expression of individual cells unit interval (Wurm, FM. (2004) Nat Biotechnol
22:1393-1398).During the fermentation, there are very effect of multiple parameters integration viable cell density, such as lactic acid (Lao, MS. et
al., (1997) Biotechnol Prog
13:688-691) with ammonia (Hansen, HA. et al., (1994) Biotechnol Prog 10:121-124) metabolic wastes.
Lactic acid produces via glycolytic pathway, and it is by lactic dehydrogenase enzymatic conversion acetone acid.The toxicity of lactic acid comes from itself, and by the neutralization alkali that added of lactic acid, the interpolation of these alkali improve simultaneously osmotic pressure (Cruz,
H. et al., (2000) Enzyme Microb Technol
27:43-52).The accumulated damage cell growth of lactic acid, also can cause the reduction of Product Expression amount.
In order to reduce the side effect that lactic acid brings, people take various measures and reduce lactic acid concn.Culture medium or incubation optimization are two kinds of measures reducing lactic acid, and these methods often promote cell consumption lactic acid (Gagnon
M. et al., (2011) Biotechnol Bioeng
108:1328-1337). however, these methods are often limited to specific cell line, after changing cell line, these formula and incubation are also required to change therewith.
Genetic engineering modified another promising method being to reduce lactic acid toxic and side effects.These methods include：By overexpression GLUT5 transhipment son be used Lactose be used as primary carbon source (Wlaschin KF. et al.,
(2007) J Biotechnol 131:168-176), overexpression Bcl-2 accelerates acetone acid to enter into tricarboxylic acid cycle (Templeton N. et al., (2014) Metab Eng 25:92-102), transhipment sub- GLUT1 RNA interference reduces glucose consumption rate (Paredes C. et al., (1999) Cytotechnology
30:85-93), overexpression malic dehydrogenase II increases consumption (Chong WPK. et al., (2010) J Biotechnol of NADH
147:116-121), overexpression pyruvate carboxylase improve enter into tricarboxylic acid cycle carbon flow (Kim S. et al.,
(2007) Appl Microbiol Biotechnol 76:659-665) and lactic dehydrogenase ribozyme interference stop lactic acid generate (Kim S. et al., (2007) Appl Microbiol Biotechnol 74:152-159).In addition, two or more genes of people's joint are to reduce lactic acid to a greater extent.Such as：Lactate dehydrogenase A is used in combination knock out and bcl-2 overexpression (Jeon M. et al., (2011) Appl Microbiol Biotechnol 92:779-790), alanine aminotransferase and taurine transporter overexpression (Tabuchi H.
et al., (2013) Biotechnol Bioeng
110: 2208-15) .These effort slow down the accumulation of lactic acid to a certain extent, however, lactic acid finally still can accumulate the concentration enough to damaging cells in fed-batch culture.
Lactic acid dehydrogenase in vivo can be reversible catalysis acetone acid and lactic acid between conversion.There are LDH-A, tri- kinds of subunits of LDH-B, LDH-C in lactic acid dehydrogenase.The lactic acid dehydrogenase hypotype being made up of different subunits has different catalytic capabilities.LDH-C hypotype is mainly expressed in sexual cell, and lactic acid conversion acetone acid can be entered tricarboxylic acid cycle by the LDH-5 hypotype being made up of it.Chinese hamster ovary cell（CHO）Mainly express LDH-A hypotype with hybridoma SP2/0 cell.In SP2/0 cell, the content of LDH-A is only 28 for the content of 5800, LDH-B hypotype, does not express LDH-C hypotype.And the expression mainly expressing LDH-A hypotype in Chinese hamster ovary celI is 50 times of LDH-C, do not express LDH-B hypotype（WO2012075124）.
WO2012075124 discloses a kind of recombinant cell lines construction method of overexpression LDH-C, lactic acid can be reduced in sweat generate, improve SP2/0 cell survival rate method, but, it does not carry out culture process, the especially optimization of nutrient media components for the recombinant cell lines of overexpression LDH-C.
Acetone acid, lactic acid, as the important metabolite in tricarboxylic acid cycle, are also the main substrate of LDH-C enzymatic reaction simultaneously.The present invention passes through to add LDH-C enzymatic reaction substrate in the culture medium of LDH-C overexpression reconstitution cell（Sodium lactate, Sodium Pyruvate etc.）, it is finally reached the lactic acid consumption improving LDH-C overexpression reconstitution cell, improve reconstitution cell survival rate, the effect of anti-apoptotic ability.
Content of the invention：
In Chinese hamster ovary cell（CHO）Culture in, lactic acid is a kind of common metabolic by-product, when it accumulate to a certain extent after cell growth inhibiting, reduction expression of recombinant proteins amount.In order to reduce lactic acid, people employ a lot of gene engineering method, but have arrived fed-batch culture latter stage, and lactic acid still accumulates the concentration enough to damaging cells.
The present invention is directed to the Chinese hamster ovary celI of overexpression LDH-C, by adding enzymatic reaction substrate in the medium（Sodium Pyruvate, sodium lactate）, the culture environment of analog cell high concentration lactic acid, it has been finally reached reduction incubation lactic acid growing amount, improved the effect of cell survival rate and anti-apoptotic ability.
The invention discloses：
1st, a kind of cultural method of the Chinese hamster ovary celI for overexpression Lactate dehydrogenase C is it is characterised in that add LDH-C enzymatic reaction substrate in culture medium.
2nd, a kind of cultural method of above-mentioned Chinese hamster ovary celI for overexpression Lactate dehydrogenase C is it is characterised in that described LDH-C enzymatic reaction substrate, containing Sodium Pyruvate 0-60mM, preferably 40mM.
3rd, a kind of cultural method of above-mentioned Chinese hamster ovary celI for overexpression Lactate dehydrogenase C is it is characterised in that described LDH-C enzymatic reaction substrate, containing sodium lactate 0-60mM, preferably 40mM.
The cultural method application of above-mentioned interpolation LDH-C enzymatic reaction substrate, the application of this cultural method can reduce the generation of lactic acid in the Chinese hamster ovary celI incubation of overexpression Lactate dehydrogenase C, extend the Chinese hamster ovary celI cultivation cycle of overexpression LDH-C, improve its anti-apoptotic ability.
In a word, the present invention is directed to the Chinese hamster ovary celI of overexpression LDH-C, by being directly added into LDH-C enzymatic reaction substrate in its culture medium, as sodium lactate, Sodium Pyruvate etc., the culture environment of simulation high concentration lactic acid state, it has been finally reached lactic acid growing amount during overexpression LDH-C recombinant cell culture, improved the effect of cell survival rate and anti-apoptotic ability.Further increase using value in actual production for the overexpression LDH-C reconstitution cell.
Brief description
Fig. 1, different clone's lactic acid dehydrogenase mRNA content
Fig. 2, the cell growth status of different clone
Fig. 3, the LDH-C enzyme activity of recombinant clone LDH-C#6
Fig. 4, the impact to cell density for the overexpression LDH-C
The impact to LDH-C cytoactive of Fig. 5, overexpression
The impact that Fig. 6, overexpression LDH-C generate to lactic acid
Fig. 7, the impact to LDH-C#6 cell density for the variable concentrations sodium lactate
Fig. 8, the impact to LDH-C#6 cytoactive for the variable concentrations sodium lactate
The impact to LDH-C#6 cell density for Fig. 9,40mM sodium lactate
The impact to LDH-C#6 cytoactive for Figure 10,40mM sodium lactate
The impact to LDH-C#6 cell lactic acid accumulation for Figure 11,40mM sodium lactate
Figure 12, the impact to LDH-C#6 cell density for the variable concentrations Sodium Pyruvate
Figure 13, the impact to LDH-C#6 cytoactive for the variable concentrations Sodium Pyruvate
The impact to LDH-C#6 cell density for Figure 14,40mM Sodium Pyruvate
The impact to LDH-C#6 cytoactive for Figure 15,40mM Sodium Pyruvate
The impact to LDH-C#6 lactic acid accumulation for Figure 16,40mM Sodium Pyruvate
The impact to LDH-C#6 early apoptosis of cells for Figure 17,40mM Sodium Pyruvate
The impact to LDH-C#6 cell late apoptic for Figure 18,40mM Sodium Pyruvate
The impact to LDH-C#6 apoptosis rate for Figure 19,40mM Sodium Pyruvate.
Specific embodiment
Following examples, experimental example describe the present invention in further detail.It should be appreciated, however, that enumerating these embodiments, experimental example has been intended merely to illustration, and it is not for limiting the present invention.
Embodiment 1：The structure of LDH-C overexpressing cell system
MCF-7 SK-BR-3 (HTB-30, ATCC, Manassas, VA,
USA) it is incubated at and comprise 10% hyclone
In 1640/DMEM (Gibco) culture medium.From people SK-BR-3
Lactate dehydrogenase gene is obtained in cell (ATCC HTB30).
First, using NucleoSpin RNA extraction agent box（Macherey-nagel, Düren, Germany）Extracted total RNA, and change into the first chain cDNA, as pcr template.RCR primer is：Front end primer, 5 '-cccaagcttgccaccatgtcaactgtcaaggagca；Rear end primer, 5 '-ccgctcgagttaaaatattagatccttttgaatat.On primer, setting restriction enzyme site Xho I and Hind III is used for PCR primer to be connected to expression vector pcDNA 3.0 (Invitrogen, Carlsbad, CA).PCR primer separates on 1% agarose gel, and reclaims purpose fragment with DNA glue reclaim test kit（Sangon
Biotech, Shanghai, China), it is then attached to cloning vehicle pGEM-T (Promega, Madison, WI).The cloning vehicle building, after Xho I and Hind III enzyme action, is connected to expression vector pcDNA 3.0, obtains carrier pcDNA3.0-LDH-C.
CHO-K1
Cell (CCL-61, ATCC) is incubated in 100 ml shaking flasks, passes on once within every four days, and passing on rear density is 5.0 × 105cells/mL.Shaking table temperature is 37.0 DEG C, rotating speed 150 rpm, and gas concentration lwevel is 5%.
By transfection reagent lipofectamine 2000
(Invitrogen) transfect pcDNA3.0-LDH-C
To in CHO-K1 (ATCC CCL-61) cell.Cell after transfection is cultivated in 1640/DMEM culture medium, and adds the cell that 10% hyclone and 0.8 mg/ml G418 have transfected purpose carrier for screening, then obtains clone with limiting dilution assay.The clone that screening obtains adapts in serum-free medium, and adds 4 mM L-Glutamine and 0.8 mg/ml G418.
In order to check whether LDH-C gene expresses, using quantitative real time PCR Instrument (Prism 7500, Applied Biosystems,
Carlsbad, CA) expression of LDH-C messenger RNA is detected.The primer of quantitative fluorescent PCR is：Front end primer, 5 '-attgtcacagcaggtgcaaggcagcaggag;
Rear end primer, 5 '-caggactataatggactatggcaggaatgattga.PCR condition is：94 DEG C of degeneration 15 seconds, 60 DEG C of annealing extend 60 seconds, and period is set to 40.Beta-actin is with comparing (Chusainow J. et al., (2010) Biotechnol Bioeng 102: 1182-1196).
Lactic dehydrogenase activity measures
Take 1.0 × 107
Cell, 1,000 4 DEG C of rpm are centrifuged 5 minutes postprecipitation phosphate-buffered salts（PBS）Rinse twice, and supersound process 3 times, 10 seconds every time, it is spaced 5 seconds between supersound process twice.Suspension after supersound process is centrifuged 10 in 4 DEG C of 12000 rpm
Min, supernatant is thick zyme extract.Thick zyme extract measures protein concentration (Beyotime, Nantong, China) with BCA method.
Lactic acid dehydrogenase both can be catalyzed lactic acid and generate acetone acid it is also possible to catalysis acetone acid generates lactic acid.The lactic dehydrogenase activity of catalysis lactic acid to acetone acid reaction measures in pH
Carry out in 7.6 kaliumphosphate buffer, comprise 70 mM sodium lactates, 2 mM NAD+, reaction temperature is 25 DEG C.Catalysis acetone acid measures in pH to the lactic dehydrogenase activity that lactic acid reacts
Carry out in 7.0 kaliumphosphate buffer, comprise 2 mM acetone acid, 0.2 mM NADH, reaction temperature is 25 DEG C.The change (△ A/min) of mensuration absorbance at 340 nm, and calculate than work.In above-mentioned reaction system, total reaction volume is 1 mL, adds the thick zyme extract of 20 μ L, the optical path of cuvette is 1 cm.
Than work=△ A/min* extension rate/0.1244* protein concentration.
In messenger RNA level, #6 clone and #20 clone all have the people LDH-C of importing to express, and other clones and negative control do not express genes of interest (Fig. 1).In cell growth level, LDH-C positive colony #6 and #20 dramatically increases (p in the viable cell density of the 4th day than comparison<0.05).And, the viable cell density that #6 is cloned in the 4th day is 6.8 × 106Cells/mL, dramatically increases (p than #20 clone viable cell density now<0.05) (Fig. 2).In lactic dehydrogenase activity level, LDH-C positive colony #6 has the ratio enzyme activity compareing higher lactic acid to acetone acid reaction, and the enzyme activity being catalyzed acetone acid to lactic acid orienting response does not have significant changes (Fig. 3).Consider these and be cloned in multilevel performance, LDH-C positive #6 clone and empty carrier Transfected clones #4 are used for follow-up study.
Embodiment 2：The cell culture of LDH-C overexpressing cell system
The cultural method of LDH-C positive colony #6 cell line is as follows：Fed-batch culture is carried out in shaking flask, Initial seeding density 5.0 × 105Cells/mL, culture medium is the serum-free medium CHOM-B01 of independent development（Zhangjiang Bioisystech Co., Ltd）.After culture 72 hours, add the supplemented medium CHOM-S01 of 3% independent research（Zhangjiang Bioisystech Co., Ltd）.3rd day starts, the daily concentration of glucose measuring in cell culture fluid, and adds to 2.5
g/L .In addition, from the beginning of the 3rd day, every other day add 1 mM L-Glutamine.Daily sampling 0.5 mL is used for the analysis of cell counting and metabolite.
Cell density is measured using blood cell counting plate, distinguishes dead cell and living cells by trypan blue dye exclusion.Lactic acid in cells and supernatant and concentration of glucose are respectively using corresponding kit measurement (Jiancheng Bioengineering Institute, Nanjing, China).
The statistical analysis of data are carried out in SPSS software, and result mean+SD represents, p value is calculated by the t inspection of paired samples and obtains.P value is considered to have statistically-significant difference less than 0.05.
The present invention tests when being not added with sodium lactate or Sodium Pyruvate first in shaking flask, the performance of LDH-C overexpression.From first day to the 3rd day, the viable cell density of the clone #6 of LDH-C overexpression dramatically increased than comparison, but the difference of three days viable cell densities afterwards is not notable（Fig. 4).In addition, the motility rate of the clone #6 of LDH-C overexpression is also not significantly different from (Fig. 5) compared with the control.
LDH-C is a kind of hypotype of lactic acid dehydrogenase, and it is closely related with lactic acid metabolism.In order to study the impact expressed to lactic acid metabolism of LDH-C, we determine the lactic acid concn in culture supernatant.In fed-batch culture, lactic acid starts by the 3rd day progressively to accumulate from culture, and its concentration maintains 15 mM afterwards, and the maximum concentration of lactic acid is not higher than 20 mM (Fig. 6).Compared with the control, LDH-C overexpression does not have a significant impact lactic acid concn.
Experimental example 1：LDH-C overexpressing cell ties up to cell growth and lactic acid generation when adding sodium lactate
The mode adding sodium lactate on 3rd day in fed-batch culture in the present invention simulates high lactic acid environment, the overexpression of induction LDH-C.After adding the sodium lactate of variable concentrations in the culture fluid in recombinant clone LDH-C#6, measure the density of cell（Fig. 7）And survival rate（Fig. 8）.Test result indicate that, after adding 40 mM sodium lactates in culture medium, the most high-density of cell and activity are significantly higher than blank.
Add the impact that sodium lactate grows to LDH-C overexpressing cell for verifying further.Add 40 mM sodium lactates in the culture fluid of recombinant clone LDH-C#6, simultaneously using the host cell pcDNA importing empty carrier
3.0#4 matched group the most, in matched group, culture medium is without sodium lactate.
After adding 40 mM sodium lactates, LDH-C overexpression clone #6 dramatically increases (Fig. 9) than comparison in the viable cell density of the 1-7 days.Motility rate also has similar performance, and from the beginning of the 5th day, the motility rate that LDH-C overexpression clones #6 significantly improves than comparison, and this trend is continued until that fed-batch culture terminates (Figure 10).After 3rd day adds sodium lactate, the concentration of lactic acid improves to 55 more than mM, and lactic acid concn is gradually reduced afterwards.At the 7th day, the lactic acid concn that LDH-C overexpression clones #6 was 12.9 mM, started lactic acid from the 3rd day and consumes 40 mM.Form sharp contrast therewith, the lactic acid concn of comparison in the 7th day is 30.4 mM, starts to consume 25.8 mM (Figure 11) from the 3rd day.The 4-7 days, the lactic acid concn that LDH-C overexpression clones #6 was substantially less than comparison (p<0.05).
Experimental example 2：LDH-C overexpressing cell ties up to cell growth and lactic acid generation when adding Sodium Pyruvate
The mode adding Sodium Pyruvate on 3rd day in fed-batch culture in the present invention induces the overexpression of LDH-C.After adding the Sodium Pyruvate of variable concentrations in the culture fluid in recombinant clone LDH-C#6, measure the density of cell（Figure 12）And survival rate（Figure 13）.Test result indicate that, after adding 40 mM Sodium Pyruvates in culture medium, the most high-density of cell and activity are significantly higher than blank.
Add the impact that Sodium Pyruvate grows to LDH-C overexpressing cell for verifying further.Add 40 mM Sodium Pyruvates in the culture fluid of recombinant clone LDH-C#6, simultaneously using the host cell pcDNA importing empty carrier
3.0#4 matched group the most, in matched group, culture medium is without Sodium Pyruvate.
By adding within the 3rd day 40 mM Sodium Pyruvates in fed-batch culture, induction LDH-C overexpression is so that highest viable cell density has reached 8.50 × 106
Cells/mL, ratio the 5.43 × 10 of comparison6Cells/mL increased 56.5% (Figure 14).And, significantly improve (Figure 15) than comparison from the motility rate of 7-9 days LDH-C overexpression clone #6.After 3rd day with the addition of Sodium Pyruvate, the lactic acid concn in this clone's culture supernatant does not increase on the 4th day immediately, but is slowly increased.When the 9th day, the lactic acid concn that LDH-C overexpression clones #6 was 40.5 mM, reduces 7.4 mM (Figure 16) than comparison.From 6-9 days, the lactic acid concn that LDH-C overexpression clones #6 was substantially less than comparison (p<0.05).
Experimental example 3：The impact to Chinese hamster ovary cell apoptosis for the LDH-C overexpression
In mammalian cell culture process, apoptosis is the principal element leading to cell death.When adding sodium lactate or Sodium Pyruvate, LDH-C overexpression significantly improves the motility rate of cell.Whether the improvement in order to determine motility rate comes from the improvement of apoptosis, and we determine the apoptosis under 40 mM Sodium Pyruvate adding conditionals.
Using Alexa
Fluor 488 annexin V/ dead cell apoptosis detection kit (Invitrogen) measures apoptosis.Its process description is as follows：Take 1.0 × 105Cell, is washed with PBS and is resuspended in 500 μ L combination buffers twice afterwards.Add 5 μ L propidium iodides and 1 μ L annexin V, then cells from light incubated at room 15 minutes uses flow cytomery.
LDH-C positive colony #6 is in the ratio of early apoptosis and reduces 12.6% than comparison, and the cell proportion being in late apoptic reduce 2.0% (Figure 17,
18).Early apoptosis and non-viable apoptotic cell ratio are added as apoptosis rate (Figure 19).After LDH-C overexpression, the apoptosis rate of the 7th and 8 day significantly reduces than comparison（p<0.01）.
SEQUENCE LISTING
<110>Shanghai Zhangjiang Biological Technology Co
<120>A kind of cultural method for overexpression Lactate dehydrogenase C zooblast
<130> 2015
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 999
<212> DNA
<213>Lactate dehydrogenase C sequence
<400> 1
atgtcaactg tcaaggagca gctaattgag aagctaattg aggatgatga
aaactcccag 60
tgtaaaatta ctattgttgg aactggtgcc gtaggcatgg cttgtgctat
tagtatctta 120
ctgaaggatt tggctgatga acttgccctt gttgatgttg cattggacaa
actgaaggga 180
gaaatgatgg atcttcagca tggcagtctt ttctttagta cttcaaagat
tacttctgga 240
aaagattaca gtgtatctgc aaactccaga atagttattg tcacagcagg
tgcaaggcag 300
caggagggag aaactcgcct tgccctggtc caacgtaatg tggctataat
gaaatcaatc 360
attcctgcca tagtccatta tagtcctgat tgtaaaattc ttgttgtttc
aaatccagtg 420
gatattttga catatatagt ctggaagata agtggcttac ctgtaactcg
tgtaattgga 480
agtggttgta atctagactc tgcccgtttc cgttacctaa ttggagaaaa
gttgggtgtc 540
caccccacaa gctgccatgg ttggattatt ggagaacatg gtgattctag
tgtgccctta 600
tggagtgggg tgaatgttgc tggtgttgct ctgaagactc tggaccctaa
attaggaacg 660
gattcagata aggaacactg gaaaaatatc cataaacaag ttattcaaag
tgcctatgaa 720
attatcaagc tgaaggggta tacctcttgg gctattggac tgtctgtgat
ggatctggta 780
ggatccattt tgaaaaatct taggagagtg cacccagttt ccaccatggt
taagggatta 840
tatggaataa aagaagaact ctttctcagt atcccttgtg tcttggggcg
gaatggtgtc 900
tcagatgttg tgaaaattaa cttgaattct gaggaggagg cccttttcaa
gaagagtgca 960
gaaacacttt ggaatattca aaaggatcta
atattttaa
999
<210> 2
<211> 332
<212> PRT
<213>Lactate dehydrogenase C protein sequence
<400> 2
Met Ser Thr Val Lys Glu Gln Leu Ile Glu Lys Leu Ile Glu Asp Asp
1
5
10
15
Glu Asn Ser Gln Cys Lys Ile Thr Ile Val Gly Thr Gly Ala Val Gly
20
25
30
Met Ala Cys Ala Ile Ser Ile Leu Leu Lys Asp Leu Ala Asp Glu Leu
35
40
45
Ala Leu Val Asp Val Ala Leu Asp Lys Leu Lys Gly Glu Met Met Asp
50
55
60
Leu Gln His Gly Ser Leu Phe Phe Ser Thr Ser Lys Ile Thr Ser Gly
65
70
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80
Lys Asp Tyr Ser Val Ser Ala Asn Ser Arg Ile Val Ile Val Thr Ala
85
90
95
Gly Ala Arg Gln Gln Glu Gly Glu Thr Arg Leu Ala Leu Val Gln Arg
100
105
110
Asn Val Ala Ile Met Lys Ser Ile Ile Pro Ala Ile Val His Tyr Ser
115
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125
Pro Asp Cys Lys Ile Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr
130
135
140
Tyr Ile Val Trp Lys Ile Ser Gly Leu Pro Val Thr Arg Val Ile Gly
145
150
155
160
Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Ile Gly Glu
165
170
175
Lys Leu Gly Val His Pro Thr Ser Cys His Gly Trp Ile Ile Gly Glu
180
185
190
His Gly Asp Ser Ser Val Pro Leu Trp Ser Gly Val Asn Val Ala Gly
195
200
205
Val Ala Leu Lys Thr Leu Asp Pro Lys Leu Gly Thr Asp Ser Asp Lys
210
215
220
Glu His Trp Lys Asn Ile His Lys Gln Val Ile Gln Ser Ala Tyr Glu
225
230
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240
Ile Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val
245
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255
Met Asp Leu Val Gly Ser Ile Leu Lys Asn Leu Arg Arg Val His Pro
260
265
270
Val Ser Thr Met Val Lys Gly Leu Tyr Gly Ile Lys Glu Glu Leu Phe
275
280
285
Leu Ser Ile Pro Cys Val Leu Gly Arg Asn Gly Val Ser Asp Val Val
290
295
300
Lys Ile Asn Leu Asn Ser Glu Glu Glu Ala Leu Phe Lys Lys Ser Ala
305
310
315
320
Glu Thr Leu Trp Asn Ile Gln Lys Asp Leu Ile Phe
325
330
Claims (3)
1. a kind of cultural method of the Chinese hamster ovary celI for overexpression Lactate dehydrogenase C is it is characterised in that add LDH-C enzymatic reaction substrate in culture medium.
2. according to claim 1, a kind of cultural method of the Chinese hamster ovary celI for overexpression Lactate dehydrogenase C is it is characterised in that described LDH-C enzymatic reaction substrate, containing Sodium Pyruvate 0-60mM.
3. according to claim 1, a kind of cultural method of the Chinese hamster ovary celI for overexpression Lactate dehydrogenase C is it is characterised in that described LDH-C enzymatic reaction substrate, containing sodium lactate 0-60mM.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349614A (en) * | 2018-12-24 | 2020-06-30 | 上海迈泰君奥生物技术有限公司 | Method for improving culture density of CHO cells |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1242440A2 (en) * | 1999-12-30 | 2002-09-25 | Specialty Assays, Inc. | Use of nicotinamide adenine dinucleotide (nad) and nicotinamide adenine dinucleotide phosphate (nadp) analogs to measure enzyme activities, metabolites and substrates |
-
2015
- 2015-07-28 CN CN201510450000.6A patent/CN106381287A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1242440A2 (en) * | 1999-12-30 | 2002-09-25 | Specialty Assays, Inc. | Use of nicotinamide adenine dinucleotide (nad) and nicotinamide adenine dinucleotide phosphate (nadp) analogs to measure enzyme activities, metabolites and substrates |
Non-Patent Citations (2)
| Title |
|---|
| 刘娜: "利用细胞工程技术改造重组蛋白生产用CHO细胞的研究进展", 《中国医药生物技术》 * |
| 靖钰: "过表达人LDH-C的CHO细胞株构建及其对乳酸代谢和细胞凋亡影响的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111349614A (en) * | 2018-12-24 | 2020-06-30 | 上海迈泰君奥生物技术有限公司 | Method for improving culture density of CHO cells |
| CN111349614B (en) * | 2018-12-24 | 2024-02-06 | 上海迈泰君奥生物技术有限公司 | Method for improving culture density of CHO cells |
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