CN107904257A - A kind of method for increasing Chinese hamster ovary celI group's expressing quantity - Google Patents

A kind of method for increasing Chinese hamster ovary celI group's expressing quantity Download PDF

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CN107904257A
CN107904257A CN201711247666.7A CN201711247666A CN107904257A CN 107904257 A CN107904257 A CN 107904257A CN 201711247666 A CN201711247666 A CN 201711247666A CN 107904257 A CN107904257 A CN 107904257A
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lactic acid
plasmid
cell
shrna
chinese hamster
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CN107904257B (en
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李鹃
张峥
蔡洁行
周伟昌
陈智胜
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Shanghai Yaoming Biomedical Co.,Ltd.
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Wuxi Biologics Shanghai Co Ltd
Wuxi Apptec Biopharmaceuticals Co Ltd
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Abstract

The present invention discloses a kind of method for increasing Chinese hamster ovary celI group's expressing quantity, it utilizes the transcription of key enzyme on shRNA technical controlling lactic acid metabolism paths, and specific steps include：1) plasmid of the structure containing protein sequence to be expressed and the plasmid containing the shRNA of key enzyme on expression lactic acid metabolism path；2) plasmid of structure in step 1) is introduced into Chinese hamster ovary celI by the method for transfection；3) antibiotic-screening is carried out to Transfected cells in step 2)；4) after cell state recovery, the cell productive target albumen after being screened using step 3)；Key enzyme includes lactic dehydrogenase, pyruvic dehydrogenase kinase on the expression lactic acid metabolism path.Method provided by the present invention needs to carry out pH the not open close addition alkali of crossing of monitoring closely come compared with adjusting in traditional Chinese hamster ovary celI all living creatures produces, the invention only additionally transfects several shRNA plasmids jointly at the same time in transfection, not only complicated degree is greatly reduced, and has larger guarantee to lactic acid reduction and then final protein yield raising.

Description

A kind of method for increasing Chinese hamster ovary celI group's expressing quantity
Technical field
The present invention relates to field of biological pharmacy, more particularly to using CHO (Chinese Hamster Ovary, Chinese storehouse Mouse gonad cell) cell expressing protein method.
Background technology
With flourishing for pharmaceutical industry, the demand of extensive Chinese hamster ovary celI protein production increasingly increases.Cell mass batch Feed supplement can provide within a short period of time gram level above albumen be used for research and develop etc. purposes, therefore its apply it is more and more extensive.Batch During feed supplement, various metabolic wastes are also constantly accumulated in cultivating system, cause cell peripheral environment to run down, and cell is lived Rate reduces, and causes the reduction of cell protein specific production rate, directly affects albumen ultimate output.
Lactic acid has a significant impact cell culture system pH as a kind of main metabolic accessory substance.Excessive lactic acid accumulation Whole system pH can be caused to be greatly reduced and then influence expression of cellular proteins.In the bioreactor, pH usually can by Line monitors and add as base to be controlled effectively, but the addition for crossing polybase can cause the increase of cultivating system osmotic pressure, together Sample is unfavorable for expression of cellular proteins.Expressed for more common shaking flask, pH can not be monitored in real time, and lactic acid is accumulated to protein expression Influence it is more notable.Lactic acid packing phenomenon can be alleviated by the precise controlling of technique sometimes, but need to expend more Manpower and materials.
The content of the invention
The technical problems to be solved by the invention are, there is provided a kind of method for increasing Chinese hamster ovary celI group's expressing quantity, its Lactic acid packing phenomenon during protein expression can be effectively reduced, without by monitoring pH and add aing base to control metabolic by-product The amount of lactic acid, reduces the pH monitoring in protein manufacturing process and adjusts operation, reduce manpower and materials.
In order to solve the above technical problems, technical solution provided by the invention is：One kind increase Chinese hamster ovary celI group's expressing quantity Method, it is using the transcription of key enzyme on shRNA technical controlling lactic acid metabolism paths, so that reaching reduces lactic acid production to carry High target protein expression quantity；
Specific steps include：
1) plasmid and contain the shRNA of key enzyme on expression lactic acid metabolism path that structure contains protein sequence to be expressed Plasmid；
2) plasmid of structure in step 1) is introduced into Chinese hamster ovary celI by the method for transfection；
3) antibiotic-screening is carried out to Transfected cells in step 2)；
4) after cell state recovery, the cell productive target albumen after being screened using step 3).
In mammalian cell, many factors can influence the metabolism of lactic acid, for example tricarboxylic acid cycle neutralizes in culture medium The concentration of lactic acid metabolism associated products, such as the concentration of sugar and acetone hydrochlorate.Key enzyme on heretofore described lactic acid metabolism path Including lactic dehydrogenase；Lactic dehydrogenase (LDH) catalysis acetonate is converted into lactic acid, controls the expression of lactic dehydrogenase can be with Directly or indirectly reduce the metabolism of lactic acid.Key enzyme swashs including pyruvic dehydrogenase on heretofore described lactic acid metabolism path Enzyme；Lactic acid can be converted into acetyl coenzyme A by pyruvic dehydrogenase, and its activity can be suppressed by pyruvic dehydrogenase kinase (PDK), The expression of control pyruvic dehydrogenase kinase can also directly or indirectly reduce the metabolism of lactic acid.
The technical solution provided in the present invention is included by suppressing in lactic dehydrogenase, pyruvic dehydrogenase kinase at least It is a kind of so as to reach reduce lactic acid effect.
In the step 1), the plasmid containing the shRNA of key enzyme on expression lactic acid metabolism path is will to express lactic acid generation Thank to the transcription original paper such as the shRNA sequences on path corresponding to key enzyme and corresponding promoter to build to plasmid.Preferably, The plasmid also is used to screen comprising antibiotic resistance gene.Destination protein gene order can be built to other and contain antibiotic resistance gene On plasmid, the plasmid containing protein sequence to be expressed is obtained.
Preferably, in the step 1), Plasmid DNA is extracted using endotoxic plasmid extraction kit is removed.
Preferably, in the step 2), shRNA plasmids and destination protein sequence can will be contained with liposome or electric shock mode Body of listing is transfected into host cell jointly.
Preferably, in the step 2), host CHO cell to be transfected on the day of transfection cell density 1 × 106Cell/ It is more than milliliter.
Preferably, in the step 3), it is interior when 5-48 is small after transfection, added into Transfected cells containing screening antibiotic Secondary culture base.
Preferably, in the step 3), after adding antibiotic-screening, every two to four days, cell change liquid or Passage.
Preferably, in the step 4), start target protein production after Cell viability recovers to more than 90%.
Preferably, in the step 4), the cell after step 3) is screened is diluted into the basal medium of production and application In, pass through the process expression target protein such as fed-batch.
The present invention also provides a kind of Chinese hamster ovary celI group of expressing protein, it is by increase Chinese hamster ovary celI group provided by the invention The method of expressing quantity obtains.
Involved shRNA (short hairpin RNA, ShorthairpinRNA) is a kind of artificial synthesized tool in the present invention There is the RNA of hairpin structure, the expression of specific gene can be suppressed by RNA interference.The present invention is directed to lactic dehydrogenase and third Ketoacid dehydrogenase kinases designs shRNA, by the plasmid containing the shRNA and albumen to be expressed when Chinese hamster ovary celI group builds Plasmid together transfect into host cell, after cell mass recovery after pass through fed-batch the methods of carry out protein production when, The shRNA will disturb the expression of the lactic dehydrogenase and/or pyruvic dehydrogenase kinase specific gene of Chinese hamster ovary celI, not only may be used Effectively to control the content of lactic acid, and the raising to protein yield has huge help.
Method provided by the present invention, is to suppress lactic acid metabolism key gene using shRNA, effectively controls Chinese hamster ovary celI The accumulation of lactic acid, significantly improves Chinese hamster ovary celI group's expressing quantity in group's protein production.Needed in being produced with traditional Chinese hamster ovary celI all living creatures PH monitor and constantly compare to adjust by adding alkali closely, the invention is only additionally common at the same time in transfection to be turned Several shRNA plasmids are contaminated, not only complicated degree is greatly reduced, and has to lactic acid reduction and then final protein yield raising Larger guarantee, therefore can have a wide range of applications in Chinese hamster ovary celI all living creatures produces.
Brief description of the drawings
Fig. 1 is that Chinese hamster ovary celI group trains under different shRNA process conditions through fed-batch different time in method of the invention Support the change curve of lactic acid concn in supernatant；1 antibody of expressing cho cell humanized IgG in A, expressing cho cell mouse source in B IgG2a antibody.
Fig. 2 is that Chinese hamster ovary celI group is thin through fed-batch different time under different shRNA process conditions in method of the invention The change curve of born of the same parents' motility rate；1 antibody of expressing cho cell humanized IgG in A, expressing cho cell mouse IgG 2a antibody in B.
Fig. 3 is that Chinese hamster ovary celI group lives under different shRNA process conditions through fed-batch different time in method of the invention The change curve of cell number；1 antibody of expressing cho cell humanized IgG in A, expressing cho cell mouse IgG 2a antibody in B.
Fig. 4 be the present invention method in Chinese hamster ovary celI group under different shRNA process conditions through different time fed-batch the Ten days and fortnight expressing quantity block diagram；1 antibody of expressing cho cell humanized IgG in A, expressing cho cell mouse source in B IgG2a antibody.
Embodiment
Clear, complete description is carried out to technical scheme below in conjunction with attached drawing, it is clear that described implementation Example is the part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this area Art personnel all other embodiments obtained on the premise of creative work is not made, belong to the model that the present invention protects Enclose.
Without special instruction, reagent and material used in implementation below are commercial prod, or are adopted Directly it is made by ordinary skill in the art means with commercial prod.
A kind of method for increasing Chinese hamster ovary celI group's expressing quantity provided by the present invention, specifically carries out according to the following steps.
Step 1, will distinguish for the shRNA of lactic dehydrogenase (LDHa) and for the shRNA of pyruvic dehydrogenase kinase Build into different plasmids.Extracted respectively containing shRNA and expression target protein using endotoxic plasmid extraction kit is removed Plasmid, target protein includes 1 antibody of humanized IgG and mouse IgG 2a antibody.
Step 2, by plasmid transfection into cell density be 106In Chinese hamster ovary celI more than cells/ml, the plasmid includes Following difference plasmid combinations, will shake pipe and put back in shaking table after mixing.Two kinds of target eggs of 1 antibody of humanized IgG and mouse IgG 2a antibody It is divided to two groups to be tested in vain.
Different condition plasmid component when table 1 transfects
Step 4, it is interior when 5 to 48 is small after transfection, the secondary culture containing corresponding screening antibiotic is added into every pipe successively Base.
Step 5, after adding screening antibiotic, passed on every 2~4 days using the secondary culture base containing screening antibiotic, Inoculum density needs to make corresponding adjustment according to Cell viability and generation time.
Step 6, when Cell viability recovers to 90% or so, cell is diluted into production according to unified inoculum density makes In basal medium, cell is put into shaking table after fully mixing.
Step 7, according to cell growth condition, suitable supplemented medium is selected to carry out fed-batch according to different cells Experiment.During fed-batch, lactic acid, Cell viability, viable count and target protein expression quantity are monitored.
Result of the test shows, 1) method of the present invention can effectively reduce lactate level.Addition, which contains, is directed to lactic dehydrogenase The lactate level of the cell mass of enzyme or pyruvic dehydrogenase kinase shRNA plasmids (LDHa groups, PDK3 groups, LDHa＆PDK3 groups) from Fed-batch mid-term begins with significant decline, and gradual with collating condition (Control groups, scramble shRNA groups) gap Increase (as shown in Figure 1).In the A of Fig. 1 at 14 days, the lactic acid concn of collating condition (Control groups, scramble shRNA groups) For 2.0-2.4 g/l, and the lactic acid concn of LDHa groups, PDK3 groups, LDHa＆PDK3 groups is 0.2-0.5 g/l, lactic acid concn phase Than have dropped 75%~92%；In the B of Fig. 1 at 14 days, the lactic acid of collating condition (Control groups, scramble shRNA groups) Concentration is 2.5-2.8 g/l, and the lactic acid concn of LDHa groups, PDK3 groups, LDHa＆PDK3 groups is 0.2-1.4 g/l, and lactic acid is dense Degree is compared and have dropped 50%~93%, wherein the biggest drop of PDK3 groups.
2) method of the invention can effectively lift Cell viability.
Addition contains cell mass (LDHa groups, the PDK3 for lactic dehydrogenase or pyruvic dehydrogenase kinase shRNA plasmids Group, LDHa＆PDK3 groups) Cell viability (Fig. 2) also increase compared with collating condition.In the A of Fig. 2 at 14 days, collating condition The Cell viability of (Control groups, scramble shRNA groups) is about 85%, and LDHa groups, PDK3 groups, LDHa＆PDK3 groups Cell viability reaches 90%, and Cell viability is compared and improves 5%；In the B of Fig. 1 at 14 days, collating condition (Control groups, Scramble shRNA groups) Cell viability be 70%, and the Cell viability of LDHa groups, PDK3 groups, LDHa＆PDK3 groups is 80- 85%, Cell viability is compared and improves 10-15%.
3) method of the invention can effectively lift viable count.
Addition contains cell mass (LDHa groups, the PDK3 for lactic dehydrogenase or pyruvic dehydrogenase kinase shRNA plasmids Group, LDHa＆PDK3 groups) viable count (Fig. 3) also increase compared with collating condition.In the A of Fig. 3 at 14 days, collating condition The viable count of (Control groups, scramble shRNA groups) is million cells/mls of 16-17, and LDHa groups, PDK3 groups, The Cell viability of LDHa＆PDK3 groups can reach more than 20,000,000 cells/mls, and viable count is compared and improves 18-25%；Fig. 1 B in 14 days when, the viable count of collating condition (Control groups, scramble shRNA groups) is 14,000,000 cells/mls, And the Cell viability of LDHa groups, PDK3 groups, LDHa＆PDK3 groups is million cells/mls of 16-17, Cell viability is compared and improved 14-21%.
4) method of the invention can effectively lift expressing quantity.
Addition contains cell mass (LDHa groups, the PDK3 for lactic dehydrogenase or pyruvic dehydrogenase kinase shRNA plasmids Group, LDHa＆PDK3 groups) expressing quantity (Fig. 4) significantly improved than collating condition.In the A of Fig. 4 at 14 days, collating condition The expressing quantity of (Control groups, scramble shRNA groups) is about 2.3 g/l, and LDHa groups, PDK3 groups, LDHa＆ The expressing quantity of PDK3 groups reaches 3.0-3.2 g/l, and expressing quantity ratio improves 30-40%；In the B of Fig. 1 at 14 days, The expressing quantity of collating condition (Control groups, scramble shRNA groups) is about 0.40-0.44 g/l, and LDHa groups, PDK3 groups, the expressing quantity of LDHa＆PDK3 groups are about 0.64-0.85 g/l, and expressing quantity ratio improves 45-112%, The lifting amplitude of wherein PDK3 groups is maximum.
Therefore, method of the invention realizes the accumulation of control cell mass lactic acid during protein expression using shRNA, Cell viability and viable count are lifted, improves cell mass state, realizes being obviously improved for target protein yield.
In conclusion the various embodiments described above and attached drawing are only presently preferred embodiments of the present invention, not limiting this The protection domain of invention, within the spirit and principles of the invention, any modification, equivalent substitution, improvement and etc. done, all should Comprising within the scope of the present invention.

Claims (12)

  1. A kind of 1. method for increasing Chinese hamster ovary celI group's expressing quantity, it is characterised in that utilize shRNA technical controlling lactic acid metabolisms The transcription of key enzyme, specific steps include on path：
    1) plasmid of the structure containing protein sequence to be expressed and the matter containing the shRNA of key enzyme on expression lactic acid metabolism path Grain；
    2) plasmid of structure in step 1) is introduced into Chinese hamster ovary celI by the method for transfection；
    3) antibiotic-screening is carried out to Transfected cells in step 2)；
    4) after cell state recovery, the cell productive target albumen after being screened using step 3)；
    Key enzyme includes lactic dehydrogenase, pyruvic dehydrogenase kinase on the expression lactic acid metabolism path.
  2. 2. the method as described in claim 1, it is characterised in that by suppressing in lactic dehydrogenase, pyruvic dehydrogenase kinase It is at least one so as to reach reduce lactic acid effect.
  3. 3. the method as described in claim 1, it is characterised in that in the step 1), closed containing expressing on lactic acid metabolism path The plasmid of the shRNA of key enzyme is by the shRNA sequences expressed on lactic acid metabolism path corresponding to key enzyme and starts accordingly On son structure to plasmid.
  4. 4. method as claimed in claim 3, it is characterised in that the plasmid also is used to screen comprising antibiotic resistance gene.
  5. 5. the method as described in claim 1, it is characterised in that in the step 1), tried using endotoxic plasmid extraction is gone Agent box extracts Plasmid DNA.
  6. 6. the method as described in claim 1, it is characterised in that, will using liposome or electric shock mode in the step 2) It is transfected into jointly containing the plasmid of shRNA of key enzyme and the plasmid containing protein sequence to be expressed on expression lactic acid metabolism path In host cell.
  7. 7. the method as described in claim 1, it is characterised in that in the step 2), host CHO cell to be transfected is worked as in transfection Its cell density is 1 × 106It is more than cells/ml.
  8. 8. the method as described in claim 1, it is characterised in that it is interior when 5-48 is small after transfection in the step 3), to after transfection The secondary culture base containing screening antibiotic is added in cell.
  9. 9. the method as described in claim 1, it is characterised in that in the step 3), after adding antibiotic-screening, arrived every two Four days, cell is carried out to change liquid or passage.
  10. 10. the method as described in claim 1, it is characterised in that in the step 4), treat that Cell viability recovers to more than 90% After start target protein production.
  11. 11. the method as described in claim 1, it is characterised in that in the step 4), the cell after step 3) is screened dilutes Into in the basal medium of production and application, pass through the process expression target protein such as fed-batch.
  12. It is 12. a kind of by Chinese hamster ovary celI group that such as claim 1-11 any one of them method is obtained.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102985437A (en) * 2010-05-28 2013-03-20 弗·哈夫曼-拉罗切有限公司 Decreasing lactate level and increasing polypeptide production by downregulating the expression of lactate dehydrogenase and pyruvate dehydrogenase kinase
CN104383557A (en) * 2014-10-16 2015-03-04 江西农业大学 Preparation method and application of RNA interfering medicine for treating pulmonary hypertension syndrome and efficacy validation method thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102985437A (en) * 2010-05-28 2013-03-20 弗·哈夫曼-拉罗切有限公司 Decreasing lactate level and increasing polypeptide production by downregulating the expression of lactate dehydrogenase and pyruvate dehydrogenase kinase
CN104383557A (en) * 2014-10-16 2015-03-04 江西农业大学 Preparation method and application of RNA interfering medicine for treating pulmonary hypertension syndrome and efficacy validation method thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
MEIXIA ZHOU等: "Decreasing lactate level and increasing antibody production in Chinese Hamster Ovary cells (CHO) by reducing the expression of lactate dehydrogenase and pyruvate dehydrogenase kinases", 《JOURNAL OF BIOTECHNOLOGY》 *
刘桂林等: "《生物技术概论》", 30 September 2010, 中国农业大学出版社 *
韩阳等: "重组蛋白的CHO细胞瞬时表达体系的研究进展", 《药物生物技术》 *

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