CN103483423B - Artificially synthesized signal peptide and application thereof - Google Patents
Artificially synthesized signal peptide and application thereof Download PDFInfo
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Abstract
The invention relates to an artificially synthesized signal peptide and application thereof. The amino acid sequence of the signal peptide is SEQ ID No, 3. The signal peptide can be added into an expression vector of a mammalian cell for guiding secretion and expression of a mammal-origin protein and is capable of increasing the expression level of an exogenous protein in animal cells.
Description
This case is the divisional application of following patent application:
Application number: 201210118230.9;
The applying date: on April 20th, 2012;
Denomination of invention: a kind of signal peptide of synthetic and application thereof
Technical field
The present invention relates to biological technical field, being specifically related to signal peptide and application thereof for guiding Mammals derived protein secreting, expressing in mammalian cell.
Technical background
The high expression of foreign gene in host cell is the prerequisite of the structure and function analysis of protein, protein or polypeptide drug research and development.Expression system for expressing recombinant protein has microorganism, plant, yeast, insect cell and zooblast etc.Mammalian cell expresses the best host with natural radioactivity albumen, it is advantageous that correctly effectively can identify eukaryotic protein synthesis, processing and secretion signal, identify and remove the intron in gene, ripe mRNA is become again through shearing, glycosylation, phosphorylation can be completed exactly, formed in chain and the post translational processing such as interchain disulfide bond and proteolysis process, thus the complete antibody produced is the same with natural antibody, there is biologic activity, not only identifiable design antigen but also can activating complement system.The DNA transfection that mammalian cell is easily reorganized, can obtain the cell transformed through screening, have genetic stability and repeatability, the product secretion of expression is easy to purifying in substratum.Mammalian cell expression protein product is utilized 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.But zooblast exogenous protein expression efficiency is low, and cost is high, therefore improve zooblast exogenous protein expression efficiency, reduction production cost is the most important thing in work at present.
Affect the factor of exogenous protein in mammalian cell a lot, as signal peptide, transcribe and translate controlling elements, RNA processing (RNA Processing), the number of gene copy, the stability of mRNA, foreign gene integration site on chromosome, recombinant protein to the genome Preference (genetic properties) etc. of the genotoxic potential of host cell and host cell.When expression vector is determined, the selection of signal peptide just seems most important.
The N-end of secretory protein is made up of the leader sequence of the 15-30Aa that can be sheared usually, this order is called signal peptide (signal sequence), at N-end or have 2-3 polarity Aa near N end place, and be all a unique hydrophobic core at the middle part of signal peptide or be made up of a lot of hydrophobic Aa.Other conservative order is not had not have acidic-group in signal peptide yet.With to lead peptide similar, signal peptide is also to be enough to any additional polypeptide to be transported into target film.Such as signal peptide is added in the N-end of globin, it just can be made no longer to stay in cytosol, and be through film and be secreted into outside born of the same parents.
Signal peptide can make the rrna translated be attached on RER film.Rrna is adhered to by the function of signal peptide and synthesis secretion albumen.Therefore own also indifference between free rrna and film binding ribosomal body.Signal peptide is as a kind of Signal analysis be attached on ER film, this can by the N-termination starting to synthesize several amino acid whose hydrophobic function.Then protein chain injects in film, and the signal peptide a kind of proteolytic enzyme be embedded in film is sheared at this moment rrna and completed translation, and albumen is along leading 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 the secernment efficiency of protein.
3) signal peptide pilot protein matter different zones or different organoid in cell can carry out correct location.
4) signal peptide can secrete the secretion that enhanser strengthens foreign protein.
5) the glad molecule being conducive to reducing expression vector of short signal is put, and improves the stability and the transcriptional efficiency that are incorporated into external source expression casette on host chromosome.
For secretory protein, select suitable signal peptide its expression amount can be significantly improved, no matter for industrial production or scientific research, its meaning is all very far-reaching.
Summary of the invention
The object of the invention is to provide a kind of signal peptide and the application thereof that can guide external source mammalian cell protein high expression, to reduce the production cost of Mammals derived Protein.
First, the invention provides a kind of signal peptide for guiding Mammals derived protein secreting, expressing in mammalian cell of synthetic, it is arbitrary that the aminoacid sequence of described signal peptide is selected from SEQ ID NO:1-5.Wherein, the signal peptide of SEQ ID NO:1-5 has higher homology.
SEQ ID NO:1MDVLAFLLGLLLLWLPGVRC
SEQ ID NO:2MDVPAEFLGLLLLWLSGVRC
SEQ ID NO:3MRVLPEFLGLLLLWISGVRC
SEQ ID NO:4MDVPLQLLGLLLLWLSGVRC
SEQ ID NO:5MDVPAELLGLLLLWISGVRC
Described mammalian cell can be CHO, BHK, SP2/0, C127 etc.
Signal peptide of the present invention is used in mammalian host cell the secreting, expressing guiding Mammals derived Protein.
Present invention also offers a kind of polynucleotide, for guiding the signal peptide of Mammals derived protein secreting, expressing in mammalian cell described in described polynucleotide encoding.
Further, what the sequence of described many nucleic acids was selected from SEQ ID NO:6-10 is arbitrary:
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 are prepared by the synthetic method of routine.
Polynucleotide of the present invention can make an addition to mammalian cell expression vector, for guiding the secreting, expressing of Mammals derived Protein.
The method of signal peptide of the present invention secreting, expressing Mammals derived Protein in zooblast is utilized to be: the polynucleotide of the polynucleotide of code book invention signal peptide and coding being expressed mammalian cell derived Protein are connected rear clone and enter mammalian cell expression vector, then express target protein by after this recombinant mammalian cells expression vector transfection mammalian cell.
Present invention also offers a kind of mammalian cell expression vector, described expression vector contains the polynucleotide of the polynucleotide of afore-mentioned code signal peptide of the present invention and the protein in encoding mammalian source.
In described expression vector, the polynucleotide front end of the protein that the polynucleotide of code book invention signal peptide are originated immediately preceding described encoding mammalian.
Further, in described expression vector, the front end of the polynucleotide of described code book invention signal peptide is added with Kozak sequence.
The promotor of described expression vector can be EF-1 α promotor, CMV promoter, SV40 late promoter or SV40 early promoter etc.
As the embodiment of the present invention is enumerated, described expression vector is the pIRESneo3 of transformation, in the pIRESneo3 of described transformation, people EF-1 α promoter sequence or people EF1-HTLV promoter sequence substituted for the main immediate early promoter sequence of hCMV in pIRESneo3 plasmid.
Present invention also offers a kind of mammalian host cell, described host cell is the transfection of foregoing expression vectors institute.Described mammalian cell can be selected from CHO, BHK, SP2/0, C127 etc.
The present invention still further provides the method for producing protein in a kind of Mammals source, for under the condition of the protein in the described Mammals source of applicable expression, cultivate aforementioned mammal host cell, from culture, then adopt ordinary method to isolate the protein in described Mammals source.
The protein in described Mammals source can be the protein of any molecular weight, as various pharmaceutical protein: erythropoietin, various there is erythropoietin activity change structure erythropoietin, and antibody etc.
Signal peptide of the present invention and protein native signal peptide compare by the present invention, the measurement result of exogenous protein expression amount shows, signal peptide of the present invention is specially adapted to Chinese hamster ovary celI, compared with existing signal peptide, exogenous protein expression amount in zooblast obtains and significantly promotes, expression level improves about 2 ~ 36 times, and its application is conducive to screening high expression level monoclonal cell strain, has reduction production cost, is convenient to the advantages such as subsequent purification.
Accompanying drawing explanation
Fig. 1 shows comparing (pEF carrier) of invention signal peptide and EPO native signal peptide
Fig. 2 shows comparing (pEV carrier) of invention signal peptide and EPO native signal peptide
Embodiment
Following embodiment describes the present invention in detail, but does not limit its invention scope
Expression vector cited by the embodiment of the present invention is transformed by Plasmid pIRES neo3 to obtain.Wherein, people EF-1 α promoter sequence, people EF1-HTLV promoter sequence substituted for the main immediate early promoter of hCMV (the Human cytomegalovirus major immediate early promoter) sequence in protoplasm grain respectively.
The synthesis of embodiment 1 signal peptide
Because signal peptide is shorter, signal peptide is pressed primer synthesis.5 ' end adds NheI restriction enzyme site and Kozak sequence, and 3 ' end adding purpose gene order part, so that add this signal peptide on goal gene.
Signalase 11:
tCGGAGCTAGCCACCaTGGATGTTCTTGCTTTTCTTCTGGGCTTGCTGCTTTTGTGGCTTCCCGGGGTGAG GTGCGCCCCACCACG(SEQ ID NO:11)
Signal peptide 2:
tCGGAGCTAGCCACCaTGGATGTTCCTGCCGAATTTCTTGGATTGTTGTTGTTGTGGCTCTCCGGAGTGCG TTGCGCCCCACCACG(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 promotor
With plasmid pEF6/V5-HisA(purchased from Invitrogen company) be masterplate, people EF-1 α promoter sequence is reference, and design primers F 01/R01, carry out polymerase chain reaction, amplify people EF-1 α promoter sequence, reaction conditions is as table 1.
F01:CATACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAG（SEQ ID NO:16）
R01:ACGGCTAGCTCCGAGCTCGGTACCAAGCTTACCTAGCCA（SEQ ID NO:17）
Table 1PCR reaction conditions
Gained PCR primer and the pUC57(through SmaI process are purchased from Fermentas company) be connected, and carry out order-checking qualification, result shows that sequence is as follows:
ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGCTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGGTAAGCTTGGTACCGAGCTCGGAGCTAGC（SEQ ID NO:18）
The amplification of embodiment 3 people EF1-HTLV promotor
With plasmid pFUSE-CHIg-hG3(purchased from InvivoGen company) be masterplate, people EF1-HTLV promoter sequence is reference, and design primers F 02/R02, carry out polymerase chain reaction, amplify people EF1-HTLV promoter sequence, reaction conditions is as table 1.
F02:ACGACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGC（SEQ ID NO:19）
R02:ATCGCTAGCGTAGGCGCCGGTCACAGCT（SEQ ID NO:20）
Gained PCR primer and the pUC57(through SmaI process are purchased from Fermentas company) be connected, and carry out order-checking qualification, result shows that sequence is as follows:
ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC（SEQ ID NO:21）
Embodiment 4 is recombinated the structure of pEF expression vector
Product in embodiment 2 and pIRESneo3 plasmid are carried out SpeI/NheI double digestion be connected, obtain plasmid pEF.
By human erythropoietin gene (the GeneBank accession number: AB463610 that codon is optimized, containing native signal peptide and 5 ' end with the addition of Kozak sequence) respectively after the process of NheI/BamHI double digestion, be connected to the plasmid pEF after NheI/BamHI double digestion, the recombinant plasmid called after pEF-EPO obtained.
By signalase 11, signal peptide 2, signal peptide 3, signal peptide 4, signal peptide 5 carries out PCR splicing with erythropoietin (no signal peptide) respectively, and splicing product and plasmid pEF carry out SpeI/NheI double digestion respectively and be connected.The recombinant plasmid obtained is called after pEF-EPO1 respectively, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5.
Embodiment 5 is recombinated pEF expression vector transient transfection expression study
Adopt liposome method (lipofectamine LTX, invitrogen) by plasmid pEF-EPO, pEF-EPO1, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5 be transfected CHO-S cells respectively, detects the transient expression level of its erythropoietin.
According to lipofectamine LTX process specifications, by 3 μ g pEF-EPO, pEF-EPO1, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5 plasmid respectively transfection 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 the CO2gas incubator of 37 DEG C after 24 hours, collect nutrient solution supernatant, expression product has erythropoietin activity through qualification, utilizes ELISA method to measure its erythropoietin expression amount.The expression amount of pEF-EPO, pEF-EPO1, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5 is 3ng/ml, 73ng/ml, 100ng/ml, 62ng/ml, 85ng/ml, 110ng/ml respectively.Result shows, and signal peptide of the present invention is relative to erythropoietin native signal peptide, and under the same conditions, EPO expression amount improves 20 ~ 36 times.
Embodiment 6 is recombinated the structure of pEV expression vector
Product in embodiment 3 and pIRESneo3 plasmid are carried out SpeI/NheI double digestion be connected, obtain plasmid pEF1-HTLV.
By human erythropoietin gene (the GeneBank accession number: AB463610 that codon is optimized, containing native signal peptide and 5 ' end with the addition of Kozak sequence) respectively after the process of NheI/BamHI double digestion, be connected to the plasmid pEV after NheI/BamHI double digestion, the recombinant plasmid called after pEV-EPO obtained.
By signalase 11, signal peptide 2, signal peptide 3, signal peptide 4, signal peptide 5 carries out PCR splicing with erythropoietin (no signal peptide) respectively, and splicing product and plasmid pEV carry out SpeI/NheI double digestion respectively and be connected.The recombinant plasmid obtained is called after pEV-EPO1 respectively, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5.
Embodiment 7 is recombinated pEV expression vector transient transfection expression study
Adopt liposome method (lipofectamine LTX, invitrogen) by plasmid pEV-EPO, pEV-EPO1, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5 be transfected CHO-S cells respectively, detects the transient expression level of its erythropoietin.
According to lipofectamine LTX process specifications, by 3 μ g pEV-EPO, pEV-EPO1, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5 plasmid respectively transfection 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 the CO2gas incubator of 37 DEG C after 24 hours, collect nutrient solution supernatant, expression product has erythropoietin activity through qualification, utilizes ELISA method to measure its erythropoietin expression amount.The expression amount of pEV-EPO, pEV-EPO1, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5 is 20ng/ml, 53ng/ml, 58ng/ml, 48ng/ml, 40ng/ml, 60ng/ml respectively.Result shows, and EPO expression amount, relative to erythropoietin native signal peptide, under the same conditions, is improve 2 ~ 3 times by signal peptide of the present invention.
Claims (10)
1. the signal peptide for guiding Mammals derived protein secreting, expressing in mammalian cell of synthetic, the aminoacid sequence of described signal peptide is SEQ ID NO:3, and the protein in described Mammals source is human erythropoietin.
2. polynucleotide, signal peptide described in described polynucleotide encoding claim 1.
3. polynucleotide as claimed in claim 2, it is characterized in that, the sequence of described many nucleic acids is SEQ ID NO:8.
4. a mammalian cell expression vector, described expression vector contains the polynucleotide of the protein in polynucleotide described in Claims 2 or 3 and encoding mammalian source, and the protein in described Mammals source is human erythropoietin.
5. mammalian cell expression vector as claimed in claim 4, it is characterized in that, polynucleotide described in Claims 2 or 3 are immediately preceding the polynucleotide front end of encoding human erythropoietin.
6. mammalian cell expression vector as claimed in claim 4, it is characterized in that, further, in described expression vector, described in Claims 2 or 3, the front end of the polynucleotide of encoding human erythropoietin is added with Kozak sequence.
7. mammalian cell expression vector as claimed in claim 4, it is characterized in that, the promotor of described expression vector is selected from EF-1 α promotor, CMV promoter, SV40 late promoter or SV40 early promoter.
8. a mammalian host cell, described host cell is the transfection of claim 4-7 arbitrary described expression vector institute.
9. mammalian host cell as claimed in claim 8, it is characterized in that, described mammalian host cell is CHO, BHK, SP2/0 or C127.
10. the method for producing protein in a Mammals source, for under the condition of the protein in applicable expression Mammals source, cultivate mammalian host cell described in claim 8 or 9, from culture, then isolate the protein in described Mammals source, the protein in described Mammals source is human erythropoietin.
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| CN107083366A (en) * | 2017-05-19 | 2017-08-22 | 何向锋 | Express adoptive immunity cell of hirudin and its production and use |
| CN116102625B (en) * | 2023-03-20 | 2023-09-22 | 杭州斯达特生物科技有限公司 | Signal peptide and application thereof |
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| WO2005121333A1 (en) * | 2004-06-14 | 2005-12-22 | Novozymes A/S | Signal peptide for producing a polypeptide |
| CN101538318A (en) * | 2009-03-06 | 2009-09-23 | 中国人民解放军军事医学科学院生物工程研究所 | Signal peptide, coding genes thereof and application |
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| WO2005121333A1 (en) * | 2004-06-14 | 2005-12-22 | Novozymes A/S | Signal peptide for producing a polypeptide |
| CN101538318A (en) * | 2009-03-06 | 2009-09-23 | 中国人民解放军军事医学科学院生物工程研究所 | Signal peptide, coding genes thereof and application |
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