CN101921315A - Artificially synthesized signal peptide and application thereof - Google Patents
Artificially synthesized signal peptide and application thereof Download PDFInfo
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- CN101921315A CN101921315A CN 201010284904 CN201010284904A CN101921315A CN 101921315 A CN101921315 A CN 101921315A CN 201010284904 CN201010284904 CN 201010284904 CN 201010284904 A CN201010284904 A CN 201010284904A CN 101921315 A CN101921315 A CN 101921315A
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Abstract
The invention relates to an artificially synthesized signal peptide and application thereof. An amino acid sequence of the signal peptide is selected from any one of SEQIDNO: 1-5. The signal peptide can be added in a mammal zooblast expression vector for guiding a mammal source protein to secrete and express, and can improve the exogenous protein expression in a zooblast by 2-36 times.
Description
Technical field
The present invention relates to biological technical field, be specifically related to be used for guiding of signal peptide and the application thereof of Mammals source protein at the mammalian cell secreting, expressing.
Technical background
Foreign gene efficiently expressing in host cell is the prerequisite of proteinic 26S Proteasome Structure and Function analysis, protein or polypeptide drug research and development.The expression system that is used for express recombinant protein has microorganism, plant, yeast, insect cell and zooblast etc.Mammalian cell is to express to have the proteic best host of natural radioactivity, it is advantageous that synthetic, processing and the secretion signal that correctly to discern eukaryotic protein effectively, intron in identification and the removal gene, be processed into sophisticated mRNA through shearing again, can finish glycosylation, phosphorylation exactly, form in the chain and translation post-treatment processes such as interchain disulfide bond and proteolysis, thereby the complete antibody that produces is the same with natural antibody, have biologic activity, but not only can discern antigen but also activating complement system.The DNA transfection that mammalian cell is easily recombinated can obtain cell transformed through screening, has genetic stability and repeatability, and expressed products is secreted into and is easy to purifying in the substratum.Utilize the mammalian cell expression protein product to be widely used in biological products industry, as a large amount of preparations of virus vaccines, antibody, Interferon, rabbit, immunomodulator, hormone and somatomedin etc.Therefore but zooblast exogenous protein expression efficient is low, and the cost height, improves zooblast exogenous protein expression efficient, and reducing production costs is the most important thing in the work at present.
It is a lot of to influence the factor of exogenous protein in mammalian cell, as integration site on karyomit(e) of the stability of signal peptide, the number of transcribing and translate controlling elements, RNA processing (RNA Processing), gene copy, mRNA, foreign gene, recombinant protein to the genome preference (genetic properties) of the genotoxic potential and the host cell of host cell etc.Under the situation that expression vector has been determined, it is most important that the selection of signal peptide just seems.
The N-end of secretory protein is made up of the leader sequence of the 15-30Aa that can be sheared usually, this is called signal peptide (signal sequence) in proper order, at N-end or near N end place 2-3 polarity Aa arranged, and all be a unique hydrophobic core at the middle part of signal peptide or constitute by a lot of hydrophobic Aa.There is not other conservative order not have acidic-group in the signal peptide yet.With to lead peptide similar, signal peptide also is can be enough to any additional polypeptide is transported into target film.For example signal peptide is added in the N-end of globin, it is no longer stayed in the cytosol, but pass film and be secreted into outside the born of the same parents.
Signal peptide can make the rrna of translating be attached on the RER film.Rrna be by the function of signal peptide adhere to and synthesis secretion proteic.So and indifference between free rrna and the film binding ribosomal body own.Signal peptide is as the signal on a kind of ER of being attached to film identification, and this may be by the hydrophobic function of the N-termination several amino acid that begins to synthesize.Protein chain injects in the film then, and signal peptide is embedded in a kind of proteolytic enzyme in the film and shears at this moment rrna and finished translation, and albumen has prolonged leads peptide by way of passing film.The effect of signal peptide secretion foreign protein is as follows:
1) signal peptide can guide secretory protein or membranin to go out born of the same parents.
2) hydrophobic core of signal peptide determines proteinic secernment efficiency.
3) signal peptide can pilot protein matter in cell different zones or different organoid carry out correct positioning.
4) signal peptide can be secreted the secretion that enhanser strengthens foreign protein.
5) the glad molecule that helps reducing expression vector of Duan signal is put, and improves the stability that is incorporated into exogenous gene expression box on the host chromosome and transcribes efficient.
For secretory protein, select the appropriate signal peptide its expression amount can be significantly improved, no matter for industrial production or scientific research, its meaning all is very far-reaching.
Summary of the invention
The object of the invention provides a kind of signal peptide and application thereof that can guide external source mammalian cell albumen to efficiently express, to reduce the Mammals proteic production cost of originating.
At first, the invention provides a kind of synthetic be used for guide the signal peptide of Mammals source protein at the mammalian cell secreting, expressing, the aminoacid sequence of described signal peptide to be selected from arbitrary among the SEQ ID NO:1-5.Wherein, the signal peptide of SEQ IDNO:1-5 has higher homology.
SEQ?ID?NO：1 MDVLAFLLGLLLLWLPGVRC
SEQ?ID?NO：2 MDVPAEFLGLLLLWLSGVRC
SEQ?ID?NO：3 MRVLPEFLGLLLLWISGVRC
SEQ?ID?NO：4 MDVPLQLLGLLLLWLSGVRC
SEQ?ID?NO：5 MDVPAELLGLLLLWISGVRC
Described mammalian cell can be CHO, BHK, SP2/0, C127 etc.
Signal peptide of the present invention is used in the mammalian host cell guiding Mammals proteic secreting, expressing of originating.
The present invention also provides a kind of polynucleotide, and described polynucleotide encoding is described to be used for guiding the signal peptide of Mammals source protein at the mammalian cell secreting, expressing.
Further, the sequence of described many nucleic acids is selected from arbitrary among the SEQ ID NO:6-10:
ATGGATGTTCTTGCTTTTCTTCTGGGCTTGCTGCTTTTGTGGCTTCCCGGGGTGAGGTGC(SEQ?ID?NO：6)
ATGGATGTTCCTGCCGAATTTCTTGGATTGTTGTTGTTGTGGCTCTCCGGAGTGCGTTGC(SEQ?ID?NO：7)
ATGAGGGTTCTGCCTGAATTCCTGGGACTTTTGTTGTTGTGGATTTCCGGCGTGCGATGT(SEQ?ID?NO：8)
ATGGATGTACCACTTCAGCTTCTTGGCTTGTTGTTGCTTTGGCTTTCTGGCGTGAGATGT(SEQ?ID?NO：9)
ATGGATGTGCCTGCTGAACTTTTGGGCCTTCTTTTGTTGTGGATATCAGGAGTACGATGC(SEQ?ID?NO：10)
Polynucleotide of the present invention can be by conventional synthetic method preparation.
Polynucleotide of the present invention can make an addition to mammalian cell expression vector, are used to guide the Mammals proteic secreting, expressing of originating.
Utilize signal peptide of the present invention secreting, expressing Mammals in zooblast proteic method of originating to be: the polynucleotide of code book invention signal peptide and coding to be expressed the mammalian cell proteic polynucleotide of originating be connected rear clone and go into mammalian cell expression vector, then will express target protein behind this recombinant mammalian cells expression vector transfection mammalian cell.
The present invention also provides a kind of mammalian cell expression vector, and described expression vector contains the polynucleotide of aforementioned code book invention signal peptide and the proteinic polynucleotide in encoding mammalian source.
In the described expression vector, the polynucleotide of code book invention signal peptide are right after the proteinic polynucleotide front end in described encoding mammalian source.
Further, in the described expression vector, the front end of the polynucleotide of described code book invention signal peptide is added with the Kozak sequence.
The promotor of described expression vector can be EF-1 α promotor, CMV promotor, SV40 late promoter or SV40 early promoter etc.
Enumerate as the embodiment of the invention, the pIRESneo3 of described expression vector for transforming, among the pIRESneo3 of described transformation, people EF-1 α promoter sequence or people EF1-HTLV promoter sequence have been replaced the main immediate early promoter sequence of hCMV in the pIRESneo3 plasmid.
The present invention also provides a kind of mammalian host cell, and described host cell is the transfection of aforementioned expression vector institute.Described mammalian cell can be selected from CHO, BHK, SP2/0, C127 etc.
The present invention also further provides the method for producing protein in a kind of Mammals source, for being fit to express under the proteinic condition in described Mammals source, cultivate the aforementioned mammal host cell, then from culture, adopt ordinary method to isolate the protein in described Mammals source.
The protein in described Mammals source can be the protein of molecular weight arbitrarily, as various pharmaceutical proteins: erythropoietin, various have that erythropoietin is active to change the structure erythropoietin, and antibody etc.
The present invention compares signal peptide of the present invention and protein self signal peptide, the measurement result of exogenous protein expression amount shows, signal peptide of the present invention is specially adapted to Chinese hamster ovary celI, compare with existing signal peptide, exogenous protein expression amount in zooblast obtains significantly to promote, expression level has improved about 2～36 times, and its application helps screening the strain of high expression level monoclonal cell, has reducing production costs, is convenient to advantages such as subsequent purification.
Description of drawings
Fig. 1 shows the comparison (pEF carrier) of invention signal peptide and EPO self signal peptide
Fig. 2 shows the comparison (pEV carrier) of invention signal peptide and EPO self signal peptide
Embodiment
Following embodiment describes the present invention in detail, but its invention scope is not limited
The expression vector that the embodiment of the invention is cited is to be transformed by plasmid pIRESneo3 to obtain.Wherein, people EF-1 α promoter sequence, people EF1-HTLV promoter sequence have been replaced the main immediate early promoter of hCMV (the Humancytomegalovirus major immediate early promoter) sequence in the former plasmid respectively.
Synthesizing of embodiment 1 signal peptide
Shorter because of signal peptide, it is synthetic that signal peptide is pressed primer.5 ' end adds NheI restriction enzyme site and Kozak sequence, and 3 ' end adding purpose gene order part is so that add this signal peptide on goal gene.
Signalase 11:
TCGGAGCTAGCCACCATGGATGTTCTTGCTTTTCTTCTGGGCTTGCTGCTTTTGTGGCTTCCCGGGGTGAG GTGC
GCCCCACCACG(SEQ ID NO:11)
Signal peptide 2:
TCGGAGCTAGCCACCATGGATGTTCCTGCCGAATTTCTTGGATTGTTGTTGTTGTGGCTCTCCGGAGTGCG TTGC
GCCCCACCACG(SEQ ID NO:12)
Signal peptide 3:
TCGGAGCTAGCCACCATGAGGGTTCTGCCTGAATTCCTGGGACTTTTGTTGTTGTGGATTTCCGGCGTGCG ATGT
GCCCCACCACG(SEQ ID NO:13)
Signal peptide 4:
TCGGAGCTAGCCACCATGGATGTACCACTTCAGCTTCTTGGCTTGTTGTTGCTTTGGCTTTCTGGCGTGAG ATGT
GCCCCACCACG(SEQ ID NO:14)
Signal peptide 5:
TCGGAGCTAGCCACCATGGATGTGCCTGCTGAACTTTTGGGCCTTCTTTTGTTGTGGATATCAGGAGTACG ATGC
GCCCCACCACG(SEQ ID NO:15)
The amplification of embodiment 2 people EF-1 α promotors
With plasmid pEF6/V5-HisA (available from Invitrogen company) is masterplate, and people EF-1 α promoter sequence is reference, and design primers F 01/R01 carries out the polymerase chain reaction, amplifies people EF-1 α promoter sequence, reaction conditions such as table 1.
F01：CATACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAG(SEQ?ID?NO：16)
R01：ACGGCTAGCTCCGAGCTCGGTACCAAGCTTACCTAGCCA(SEQ?ID?NO：17)
Table 1PCR reaction conditions
Gained PCR product is connected with the pUC57 (available from Fermentas company) that handles through SmaI, and the evaluation of checking order, and the result shows that sequence is as follows:
ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGCTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGGTAAGCTTGGTACCGAGCTCGGAGCTAGC(SEQ?ID?NO：18)
The amplification of embodiment 3 people EF1-HTLV promotors
With plasmid pFUSE-CHIg-hG3 (available from InvivoGen company) is masterplate, and people EF1-HTLV promoter sequence is reference, and design primers F 02/R02 carries out the polymerase chain reaction, amplifies people EF1-HTLV promoter sequence, reaction conditions such as table 1.
F02：ACGACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGC(SEQ?ID?NO：19)
R02：ATCGCTAGCGTAGGCGCCGGTCACAGCT(SEQ?ID?NO：20)
Gained PCR product is connected with the pUC57 (available from Fermentas company) that handles through SmaI, and the evaluation of checking order, and the result shows that sequence is as follows:
ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC(SEQ?ID?NO：21)
The structure of embodiment 4 reorganization pEF expression vectors
Product among the embodiment 2 and pIRESneo3 plasmid are carried out the SpeI/NheI double digestion be connected, obtain plasmid pEF.
Human erythropoietin gene (GeneBank accession number: AB463610 with codon optimization, contain self signal peptide and 5 ' end and added the Kozak sequence) respectively after the NheI/BamHI double digestion is handled, be connected to the plasmid pEF behind the NheI/BamHI double digestion, the recombinant plasmid called after pEF-EPO that is obtained.
With signalase 11, signal peptide 2, signal peptide 3, signal peptide 4, signal peptide 5 carry out the PCR splicing with erythropoietin (no signal peptide) respectively, and splicing product carries out the SpeI/NheI double digestion respectively with plasmid pEF and is connected.The recombinant plasmid that is obtained is called after pEF-EPO1 respectively, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5.
Embodiment 5 reorganization pEF expression vector transient transfection expression studies
The employing liposome method (lipofectamine LTX, invitrogen) with plasmid pEF-EPO, pEF-EPO1, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5 be transfection CHO-S cell respectively, detects the transient expression level of its erythropoietin.
According to lipofectamine LTX process specifications, with 3 μ g pEF-EPO, pEF-EPO1, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5 plasmid transfection respectively is incubated at the CHO-S cell (about 1.1 * 10 of 6 orifice plates (2.5ml/well, 10% foetal calf serum)
6Individual cell).Transfectional cell is at 5%CO
2, cultivate in 37 ℃ the CO2gas incubator after 24 hours, collect the nutrient solution supernatant, expression product has the erythropoietin activity through evaluation, utilizes the ELISA method to measure its erythropoietin expression amount.PEF-EPO, pEF-EPO1, pEF-EPO2, pEF-EPO3, pEF-EPO4, the expression amount of pEF-EPO5 are respectively 3ng/ml, 73ng/ml, 100ng/ml, 62ng/ml, 85ng/ml, 110ng/ml.The result shows that signal peptide of the present invention is with respect to erythropoietin self signal peptide, and under the same conditions, the EPO expression amount has improved 20～36 times.
The structure of embodiment 6 reorganization pEV expression vectors
Product among the embodiment 3 and pIRESneo3 plasmid are carried out the SpeI/NheI double digestion be connected, obtain plasmid pEF1-HTLV.
Human erythropoietin gene (GeneBank accession number: AB463610 with codon optimization, contain self signal peptide and 5 ' end and added the Kozak sequence) respectively after the NheI/BamHI double digestion is handled, be connected to the plasmid pEV behind the NheI/BamHI double digestion, the recombinant plasmid called after pEV-EPO that is obtained.
With signalase 11, signal peptide 2, signal peptide 3, signal peptide 4, signal peptide 5 carry out the PCR splicing with erythropoietin (no signal peptide) respectively, and splicing product carries out the SpeI/NheI double digestion respectively with plasmid pEV and is connected.The recombinant plasmid that is obtained is called after pEV-EPO1 respectively, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5.
Embodiment 7 reorganization pEV expression vector transient transfection expression studies
The employing liposome method (lipofectamine LTX, invitrogen) with plasmid pEV-EPO, pEV-EPO1, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5 be transfection CHO-S cell respectively, detects the transient expression level of its erythropoietin.
According to lipofectamine LTX process specifications, with 3 μ g pEV-EPO, pEV-EPO1, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5 plasmid transfection respectively is incubated at the CHO-S cell (about 1.1 * 10 of 6 orifice plates (2.5ml/well, 10% foetal calf serum)
6Individual cell).Transfectional cell is at 5%CO
2, cultivate in 37 ℃ the CO2gas incubator after 24 hours, collect the nutrient solution supernatant, expression product has the erythropoietin activity through evaluation, utilizes the ELISA method to measure its erythropoietin expression amount.PEV-EPO, pEV-EPO1, pEV-EPO2, pEV-EPO3, pEV-EPO4, the expression amount of pEV-EPO5 are respectively 20ng/ml, 53ng/ml, 58ng/ml, 48ng/ml, 40ng/ml, 60ng/ml.The result shows that signal peptide of the present invention under the same conditions, has improved 2～3 times with the EPO expression amount with respect to erythropoietin self signal peptide.
Claims (13)
- A synthetic be used for guide the signal peptide of Mammals source protein at the mammalian cell secreting, expressing, the aminoacid sequence of described signal peptide to be selected from arbitrary among the SEQ ID NO:1-5.
- 2. signal peptide is used at the mammalian host cell guiding Mammals proteic secreting, expressing of originating according to claim 1.
- 3. polynucleotide, the described signal peptide of described polynucleotide encoding claim 1.
- 4. as polynucleotide as described in the claim 3, it is characterized in that the sequence of described many nucleic acids is selected from arbitrary among the SEQ ID NO:6-10.
- 5. mammalian cell expression vector, described expression vector contain the proteinic polynucleotide in claim 3 or 4 described polynucleotide and encoding mammalian source.
- 6. as mammalian cell expression vector as described in the claim 5, it is characterized in that claim 3 or 4 described polynucleotide are right after the proteinic polynucleotide front end in described encoding mammalian source.
- 7. as mammalian cell expression vector as described in the claim 5, it is characterized in that further, in the described expression vector, the front end of claim 3 or 4 described polynucleotide is added with the Kozak sequence.
- 8. as mammalian cell expression vector as described in the claim 5, it is characterized in that the promotor of described expression vector is selected from EF-1 α promotor, CMV promotor, SV40 late promoter or SV40 early promoter.
- 9. as mammalian cell expression vector as described in the claim 5, it is characterized in that the protein in described Mammals source is selected from erythropoietin, various have that erythropoietin is active to change the structure erythropoietin, and antibody.
- 10. mammalian host cell, described host cell are the transfection of the arbitrary described expression vector of claim 5-9 institute.
- 11., it is characterized in that described mammalian host cell is CHO, BHK, SP2/0 or C127 as mammalian host cell as described in the claim 10.
- 12. the method for producing protein in a Mammals source, for being fit to express under the proteinic condition in Mammals source, cultivate claim 10 or 11 described mammalian host cells, then from culture, isolate the protein in described Mammals source.
- 13. method for producing protein as Mammals source as described in the claim 12, it is characterized in that, the protein in described Mammals source is selected from erythropoietin, and various have that erythropoietin is active to change the structure erythropoietin, and antibody.
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| CN104928285A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院沈阳应用生态研究所 | Vector cloning and expression region gene and protein secretion type mammal cell expression vector |
| CN104974226A (en) * | 2014-04-01 | 2015-10-14 | 上海中信国健药业股份有限公司 | Signal peptide for protein expression |
| CN110760541A (en) * | 2019-10-31 | 2020-02-07 | 中国农业科学院兰州兽医研究所 | Selection method and application of signal peptide when Chinese hamster ovary cells express foreign proteins |
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| WO2008060941A2 (en) * | 2006-11-17 | 2008-05-22 | The Regents Of The University Of California | Expression system of nell peptide |
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| WO2008060941A2 (en) * | 2006-11-17 | 2008-05-22 | The Regents Of The University Of California | Expression system of nell peptide |
| CN101687910A (en) * | 2007-06-04 | 2010-03-31 | 英国龙沙生物医药股份有限公司 | Mammalian expression vector with a highly efficient secretory signal sequence |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104928285A (en) * | 2014-03-21 | 2015-09-23 | 中国科学院沈阳应用生态研究所 | Vector cloning and expression region gene and protein secretion type mammal cell expression vector |
| CN104974226A (en) * | 2014-04-01 | 2015-10-14 | 上海中信国健药业股份有限公司 | Signal peptide for protein expression |
| CN104974226B (en) * | 2014-04-01 | 2019-10-15 | 三生国健药业（上海）股份有限公司 | A kind of signal peptide for protein expression |
| CN110760541A (en) * | 2019-10-31 | 2020-02-07 | 中国农业科学院兰州兽医研究所 | Selection method and application of signal peptide when Chinese hamster ovary cells express foreign proteins |
| CN110760541B (en) * | 2019-10-31 | 2022-03-29 | 中国农业科学院兰州兽医研究所 | Selection method and application of signal peptide when Chinese hamster ovary cells express foreign proteins |
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