CN102659927B - Synthetic signal peptide and application thereof - Google Patents
Synthetic signal peptide and application thereof Download PDFInfo
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- CN102659927B CN102659927B CN201210118230.9A CN201210118230A CN102659927B CN 102659927 B CN102659927 B CN 102659927B CN 201210118230 A CN201210118230 A CN 201210118230A CN 102659927 B CN102659927 B CN 102659927B
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Abstract
The invention relates to a synthetic signal peptide and application thereof. The amino acid sequence of the signal peptide is one of SEQ ID NO: 3-5. The signal peptide can be added into an expression vector of a mammalian cell, is used for inducing secretion and expression of a mammalian protein and can improve the expression level of an exogenous protein in an animal cell.
Description
This case is dividing an application of following patent application:
Application number: 201010284904.3;
The applying date: on September 17th, 2010;
Denomination of invention: a kind of signal peptide of synthetic and application thereof
Technical field
The present invention relates to biological technical field, be specifically related to for guiding Mammals source protein in signal peptide and the application thereof of mammalian cell secreting, expressing.
Technical background
The high efficient expression of foreign gene in host cell is the prerequisite of structure and function analysis, protein or the polypeptide drug research and development of protein.For expressing the expression system of recombinant protein, there are microorganism, plant, yeast, insect cell and zooblast etc.Mammalian cell is to express the best host with natural radioactivity albumen, it is advantageous that synthetic, processing and the secretion signal that can correctly effectively identify eukaryotic protein, intron in identification and removal gene, through shearing, become ripe mRNA again, can complete exactly glycosylation, phosphorylation, form in chain and translate post-treatment process with interchain disulfide bond and proteolysis etc., thereby the complete antibody producing is the same with natural antibody, there is biologic activity, not only can identify antigen but also can activating complement system.Easily reorganized DNA transfection of mammalian cell, can obtain the cell that transforms through screening, have genetic stability and repeatability, and the product of expression is secreted into and in substratum, is easy to purifying.Utilize 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.But zooblast exogenous protein expression efficiency is low, and cost is high, therefore improves zooblast exogenous protein expression efficiency, and reducing production costs 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 integration site on karyomit(e) of controlling elements, RNA processing (RNA Processing), the number of gene copy, the stability of mRNA, foreign gene, recombinant protein to the genome Preference of the genotoxic potential of host cell and host cell (genetic properties) etc.In the situation that expression vector is definite, it is most important that the selection of signal peptide just seems.
The N-end of secretory protein is comprised of the leader sequence of the 15-30Aa that can be sheared conventionally, this is sequentially called signal peptide (signal sequence), at N-end or near N end place, there is 2-3 polarity Aa, and at the middle part of signal peptide, be all a unique hydrophobic core or formed by a lot of hydrophobic Aa.In signal peptide, do not have other conservative order there is no acidic-group yet.With to lead peptide similar, signal peptide is to be also enough to any additional polypeptide to be transported into target film.The N-end that for example signal peptide is added in to globin, just can make it no longer stay in cytosol, but passes film and be secreted into outside born of the same parents.
Signal peptide can make the rrna of translating be attached on RER film.Rrna is to adhere to and synthesis secretion albumen by the function of signal peptide.So and indifference between free rrna and film binding ribosomal body own.Signal peptide is as the signal identification on a kind of ER of being attached to film, this may be by the N-termination that starts to synthesize several amino acid whose hydrophobic functions.Then protein chain injects in film, and signal peptide is embedded in a kind of proteolytic enzyme in film and shears at this moment rrna and completed 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 the secernment efficiency of protein.
3) signal peptide can pilot protein matter in cell different zones or different organoid carry out correct location.
4) signal peptide can be secreted the secretion that enhanser strengthens foreign protein.
5) the glad molecule that is conducive to reduce expression vector of short signal is put, and improves and is incorporated into the stability of external source expression casette on host chromosome and transcribes efficiency.
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 application thereof that can guide the high efficient expression of external source mammalian cell albumen, carrys out the production cost of source protein to reduce Mammals.
First, the invention provides a kind of synthetic for guiding Mammals source protein at the signal peptide of mammalian cell secreting, expressing, the aminoacid sequence of described signal peptide is selected from arbitrary in SEQ ID NO:1-5.Wherein, the signal peptide of SEQ ID NO: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 mammalian host cell and guides Mammals to carry out the secreting, expressing of source protein.
The present invention also provides a kind of polynucleotide, described in described polynucleotide encoding for guiding Mammals source protein at the signal peptide of mammalian cell secreting, expressing.
Further, the sequence of described many nucleic acids is selected from arbitrary in 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 prepared by conventional synthetic method.
Polynucleotide of the present invention can make an addition to mammalian cell expression vector, for guiding Mammals to carry out the secreting, expressing of source protein.
Utilize signal peptide of the present invention secreting, expressing Mammals in zooblast to come the method for source protein to be: the polynucleotide of code book invention signal peptide and coding to be expressed to mammalian cell and come the polynucleotide of source protein to be connected rear clone to enter mammalian cell expression vector, then will after this recombinant mammalian cells expression vector transfection mammalian cell, express target protein.
The present invention also provides a kind of mammalian cell expression vector, the polynucleotide of the protein in the polynucleotide that described expression vector contains aforementioned code book invention signal peptide and encoding mammalian source.
In described expression vector, the polynucleotide front end of the protein that the polynucleotide of code book invention signal peptide are immediately originated at 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 promotor, 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 have been replaced the main immediate early promoter sequence of hCMV in 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 under the condition of protein that is applicable to the described Mammals of expression source, cultivate 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 there is erythropoietin activity change 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 and promotes, expression level has improved approximately 2～36 times, and its application is conducive to screen the strain of high expression level monoclonal cell, has reducing production costs, is convenient to the advantages such as subsequent purification.
Accompanying drawing explanation
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 present invention is cited is to be obtained by Plasmid pIRES neo3 transformation.Wherein, people EF-1 α promoter sequence, people EF1-HTLV promoter sequence have been replaced respectively the main immediate early promoter of hCMV (the Human cytomegalovirus major immediate early promoter) sequence in former plasmid.
Synthesizing of embodiment 1 signal peptide
Shorter because of signal peptide, signal peptide is pressed to primer synthetic.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 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
The plasmid pEF6/V5-HisA (purchased from Invitrogen company) of take is masterplate, and people EF-1 α promoter sequence is reference, and design primers F 01/R01, carries out polymerase chain reaction, amplifies people EF-1 α promoter sequence, and reaction conditions is 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 (purchased from Fermentas company) processing through SmaI, and the evaluation of checking order, and result shows that sequence is as follows:
ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCA
ATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCG
AGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACAC
AGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCA
CCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAG
GAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTC
GCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGC
AAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCC
GTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAG
CTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGG
CACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGG
GAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGG
AGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGT
TTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCT
TGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCAT
TTCAGGTGTCGTGAGGAATTAGCTTGGTACTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGGTAAGCTTGGT
ACCGAGCTCGGAGCTAGC(SEQ ID NO：18)
The amplification of embodiment 3 people EF1-HTLV promotors
The plasmid pFUSE-CHIg-hG3 (purchased from InvivoGen company) of take is masterplate, and people EF1-HTLV promoter sequence is reference, and design primers F 02/R02, carries out polymerase chain reaction, amplifies people EF1-HTLV promoter sequence, and reaction conditions is as table 1.
F02：ACGACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGC(SEQ ID NO：19)
R02：ATCGCTAGCGTAGGCGCCGGTCACAGCT(SEQ ID NO：20)
Gained PCR product is connected with the pUC57 (purchased from Fermentas company) processing through SmaI, and the evaluation of checking order, and result shows that sequence is as follows:
ACTAGTGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCA
ATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCG
AGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACAC
AGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGA
GTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGA
GACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTG
CTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC
(SEQ ID NO：21)
The structure of embodiment 4 restructuring pEF expression vectors
Product in embodiment 2 and pIRESneo3 plasmid are carried out to SpeI/NheI double digestion and be connected, obtain plasmid pEF.
Human erythropoietin gene (the GeneBank accession number: AB463610 that codon is optimized, containing self signal peptide and 5 ' end, added Kozak sequence) respectively after the processing of NheI/BamHI double digestion, be connected to the plasmid pEF after NheI/BamHI double digestion, the recombinant plasmid called after pEF-EPO obtaining.
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 carries out respectively SpeI/NheI double digestion with plasmid pEF and is connected.The recombinant plasmid obtaining is called after pEF-EPO1 respectively, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5.
Embodiment 5 restructuring pEF expression vector transient transfection expression studies
Adopt liposome method (lipofectamine LTX, invitrogen) by plasmid pEF-EPO, pEF-EPO1, pEF-EPO2, pEF-EPO3, pEF-EPO4, pEF-EPO5 is transfection CHO-S cell 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 (approximately 1.1 * 10 of 6 orifice plates (2.5ml/well, 10% foetal calf serum)
6individual cell).Transfectional cell is at 5%CO
2, in the CO2gas incubator of 37 ℃, cultivate after 24 hours, collect nutrient solution supernatant, expression product has erythropoietin activity through identifying, utilizes ELISA method to measure its erythropoietin expression amount.PEF-EPO, pEF-EPO1, pEF-EPO2, pEF-EPO3, pEF-EPO4, the expression amount of pEF-EPO5 is respectively 3ng/ml, 73ng/ml, 100ng/ml, 62ng/ml, 85ng/ml, 110ng/ml.Result demonstration, signal peptide of the present invention is with respect to erythropoietin self signal peptide, and under the same conditions, EPO expression amount has improved 20～36 times.
The structure of embodiment 6 restructuring pEV expression vectors
Product in embodiment 3 and pIRESneo3 plasmid are carried out to SpeI/NheI double digestion and be connected, obtain plasmid pEF1-HTLV.
Human erythropoietin gene (the GeneBank accession number: AB463610 that codon is optimized, containing self signal peptide and 5 ' end, added Kozak sequence) respectively after the processing of NheI/BamHI double digestion, be connected to the plasmid pEV after NheI/BamHI double digestion, the recombinant plasmid called after pEV-EPO obtaining.
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 carries out respectively SpeI/NheI double digestion with plasmid pEV and is connected.The recombinant plasmid obtaining is called after pEV-EPO1 respectively, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5.
Embodiment 7 restructuring pEV expression vector transient transfection expression studies
Adopt liposome method (lipofectamine LTX, invitrogen) by plasmid pEV-EPO, pEV-EPO1, pEV-EPO2, pEV-EPO3, pEV-EPO4, pEV-EPO5 is transfection CHO-S cell 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 (approximately 1.1 * 10 of 6 orifice plates (2.5ml/well, 10% foetal calf serum)
6individual cell).Transfectional cell is at 5%CO
2, in the CO2gas incubator of 37 ℃, cultivate after 24 hours, collect nutrient solution supernatant, expression product has erythropoietin activity through identifying, utilizes ELISA method to measure its erythropoietin expression amount.PEV-EPO, pEV-EPO1, pEV-EPO2, pEV-EPO3, pEV-EPO4, the expression amount of pEV-EPO5 is respectively 20ng/ml, 53ng/ml, 58ng/ml, 48ng/ml, 40ng/ml, 60ng/ml.Result demonstration, signal peptide of the present invention, with respect to erythropoietin self signal peptide, under the same conditions, has improved 2～3 times by EPO expression amount.
Claims (10)
- Synthetic for guiding Mammals source protein at a signal peptide for mammalian cell secreting, expressing, the aminoacid sequence of described signal peptide is as shown in SEQ ID NO:4, 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, is characterized in that, the sequence of described polynucleotide is as shown in SEQ ID NO:9.
- 4. a mammalian cell expression vector, the polynucleotide that described expression vector contains polynucleotide and encoding human erythropoietin described in claim 2 or 3.
- 5. mammalian cell expression vector as claimed in claim 4, is characterized in that, polynucleotide are immediately at the polynucleotide front end of described human erythropoietin described in claim 2 or 3.
- 6. mammalian cell expression vector as claimed in claim 4, is characterized in that, further, in described expression vector, the front end of polynucleotide is added with Kozak sequence described in claim 2 or 3.
- 7. mammalian cell expression vector as claimed in claim 4, 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.
- 8. a mammalian host cell, described host cell is the transfection of the arbitrary described expression vector of claim 4-7 institute.
- 9. mammalian host cell as claimed in claim 8, is characterized in that, described mammalian host cell is CHO, BHK, SP2/0 or C127.
- 10. the method for producing protein in Mammals source, for being applicable to expressing under the condition of human erythropoietin, cultivates mammalian host cell described in claim 8 or 9, then from culture, isolates described human erythropoietin.
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