CN110452288A - A kind of artificial synthesized signal peptide - Google Patents
A kind of artificial synthesized signal peptide Download PDFInfo
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- CN110452288A CN110452288A CN201810432932.1A CN201810432932A CN110452288A CN 110452288 A CN110452288 A CN 110452288A CN 201810432932 A CN201810432932 A CN 201810432932A CN 110452288 A CN110452288 A CN 110452288A
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
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- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/67—General methods for enhancing the expression
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Abstract
The invention belongs to field of biotechnology, disclose a kind of artificial synthesized signal peptide.Specifically, the present invention relates to a signal peptides for guiding foreign protein to express in mammalian cell hypersecretion, the signal peptide amino acid sequence is as shown in SEQ ID NO:6, before this signal peptide is added in the amino acid sequence of albumen, for guiding foreign protein in the secreting, expressing of mammalian cell, this signal peptide can increase substantially the expression quantity of albumen in mammalian cells compared to other convectional signals peptides.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of artificial synthesized signal peptide, in particular to it is a kind of for drawing
Foreign protein is led in the artificial synthesized signal peptide of mammalian cell secreting, expressing.
Background technique
According to thomson Reuter latest data, the protein drug that the whole world had listed or entered three phases clinic has 243,
Wherein 121 have obtained U.S. FDA approval, based on tumour and autoimmune disease medication, in ophthalmology disease, respiratory
Systemic disease, anti infection region also have the application of multiple kinds.It is expected that the scale in global protein drug market in 2018 has surpassed
Cross 100,000,000,000 dollars.
One outstanding feature of mammalian cell expression albumen is that expression is low, in recent years, with molecular biology,
The development of cell biology has also carried out a series of research to expressing quantity is improved, the design including carrier, cell strain
Transformation, the optimization of fermentation condition, fermentation medium optimization, the optimization etc. of feed profile.Wherein done in terms of vector modification
Many work, the selection including promoter appropriate, enhancer, tailing signal, signal peptide can improve CHO on certain level
The expressing quantity of cell.The expression quantity of the albumen of different signal inducing peptides is different, and suitable signal peptide is selected to can be improved
The expression quantity of albumen.
Signal peptide is that the newly synthesized protein of guidance can positioned at the N-terminal of secretory protein to the short peptide chain that secretion access shifts
It is sheared, is generally made of 15-30 amino acid leader sequence, including three areas: positively charged N-terminal has 2-3 polarity ammonia
Base acid, referred to as basic amine group end；Centre constitutes hydrophobic sequence by many hydrophobic amino acids, it is the major function of signal peptide
Area；One longer negatively charged C-terminal, is signal sequence cleavage site, also referred to as processing district.When signal peptide sequence synthesizes
Afterwards, identified by signal recognition particle (SRP), protein synthesis pause or slow down, signal recognition particle by ribosomes carry to
In endoplasmic reticulum, protein synthesis restarts, and under the guidance of signal peptide, newly synthesized protein enters endoplasmic, and believes
Number peptide sequence is then removed under the action of signal peptidase.Signal peptide is enough any additional polypeptide being transported to target membrane, different
Albumen is expressed in mammalian cells, needs different signal peptides, same albumen under the action of unlike signal peptide, even if
Can secreting, expressing, secernment efficiency can also differ greatly, select suitable signal peptide, the secreting, expressing of albumen can be improved
Amount.For secretory protein, select suitable signal peptide that can significantly improve its expression quantity, no matter for industrial production or section
Research is learned, all there is very profound significance.
Summary of the invention
The purpose of the present invention is to provide a kind of artificial synthesized signal peptide, and specifically one kind can guide foreign protein
The signal peptide of matter high efficient expression, to reduce the production cost of foreign protein.
The present invention provides a kind of artificial synthesized for guiding exogenous proteins secreting, expressing in mammalian cells
Signal peptide.For the signal peptide amino acid sequence as shown in SEQ ID NO:6, particular sequence is as follows:
MGKLIFWLVFWLTIFWLGSA。
The mammalian cell is Chinese hamster ovary cell (Chinese hamster ovary, CHO), BHK,
HEK293 cell or SP2/0 cell.
The present invention also provides a kind of external source GLP-1-Fc fusion protein, FGF21 cell factor, PD-1 antibody, anti-CD20
The method of antibody-secreting expression, which is characterized in that use the signal peptide as shown in SEQ ID NO:6.
The present invention also provides a kind of polynucleotides, for guiding foreign protein in lactation described in the polynucleotide encoding
The signal peptide of secreting, expressing in zooblast.
Further, for the polynucleotide sequence as shown in SEQ ID NO:12, particular sequence is as follows:
ATGGGCAAACTCATATTCTGGCTTGTGTTTTGGCTGACTATTTTTTGGCTTGGATCAGCT。
Polynucleotides of the invention can pass through the methods of conventional chemical synthesis preparation.
Polynucleotides of the invention can make an addition to mammalian cell expression vector, for guiding the secretion table of foreign protein
It reaches.
Utilize the method for signal peptide of the invention secreting, expressing protein in mammalian cells: will coding present invention letter
The polynucleotides of number peptide connect rear clone with the polynucleotides of coding expression protein and enter mammalian cell expression vector, then
By express express target protein after the recombinant mammalian cells expression vector transfection mammalian cell.
The present invention also provides a kind of mammalian cell expression vector, the expression vector contains the afore-mentioned code present invention
The polynucleotides of signal peptide and the polynucleotides for encoding target protein.The polynucleotides are immediately in the encoding mammalian
The polynucleotides front end of derived protein.
In one embodiment, carrier used by expressed fusion protein is Freedom pCHO1.0, synthesizes and constructs letter
Number peptide plasmid uses I double digestion of restriction enzyme A vr II and BstZ17 after extracting plasmid, carries out gel electrophoresis, recycles purpose piece
The aim sequence of signal peptide, is cloned into the restriction enzyme site of Avr II and BstZ17 I by section.Table containing signal peptide and destination protein
Up to carrier transfection host cell and express express target protein.
In another embodiment, carrier used by the expression cell factor is Freedom pCHO1.0, synthesizes and constructs
Signal peptide plasmid uses I double digestion of restriction enzyme A vr II and BstZ17 after extracting plasmid, carries out gel electrophoresis, recycles purpose
The aim sequence of signal peptide is cloned into the restriction enzyme site of Avr II and BstZ17 I by segment.Containing signal peptide and destination protein
Expression vector transfection host cell, and express express target protein.The carrier can also be PXC17.4, pCDNA3.3, pCMV3
Equal expression vectors, express express target protein effect are close with carrier Freedom pCHO1.0.
In another embodiment, expressing carrier used in antibody is pCDNA3.3, merges and construct plasmid containing signal peptide,
After sequencing identification, expression vector transfection host cell, and express express target protein.
The promoter of the expression vector is EF-1 α promoter, hCMV promoter, SV40 promoter or EF-1 α and hCMV
The hybrid promoter of promoter.
The present invention also provides a kind of mammalian host cell, the host cell is transfected by foregoing expression vectors.
The mammalian cell can be selected from CHO, BHK, HEK293 or SP2/0.
The present invention also provides a kind of preparation methods of mammal foreign protein, are being suitble to expression mammal foreign protein
Under conditions of matter, mammalian host cell is cultivated, the mammal exogenous proteins are then isolated from culture.
Signal peptide of the invention can be used for the secreting, expressing of fusion protein, cell factor or antibody.
In a preferred embodiment, by the multicore glycosides of encoding amino acid sequence signal peptide as shown in SEQ ID NO:6
Acid connect rear clone with the polynucleotides of coding GLP-1-Fc and enters expression vector FreedompCHO1.0, then by the recombination lactation
The CHO-S cell express express target protein of animal cell expression vectors transfection Gibico company.The result shows that signal peptide of the present invention with
GLP-1-Fc fusion protein original signal peptide human albumin is compared, and signal peptide of the invention may make GLP-1-Fc
The secreting, expressing amount of fusion protein has been increased to 4813.3ug/ml from 785.8ug/ml, improves 6.12 times.Signal of the invention
Peptide can be such that the expression quantity of fusion protein increases substantially.
In one more preferably embodiment, firstly, the polynucleotides using signal peptide shown in SEQ ID NO:6 construct recombination
PCHO1.0 expression vector: being synthesized by Shanghai bioengineering Co., Ltd and constructs signal peptide plasmid puc 57-GLP-1-Fc-6, small
After amount extracts plasmid, with restriction enzyme A vr II, I double digestion of BstZ17, agarose gel electrophoresis, glue recycling are then carried out
Target fragment.By target fragment and the equally carrier for expression of eukaryon through restriction enzyme A vr II, I double digestion of BstZ17
PCHO1.0 connection constructs recombinant eukaryon expression vector pCHO 1.0-GLP-1-Fc-6, converts E. coli DH5 α,
It is incubated overnight rear picking positive colony and carries out mini-scale plasmid extraction, agar after restriction enzyme A vr II, I double digestion of BstZ17
The positive clone of identification is served the sequencing of marine growth Engineering Co., Ltd by sugared gel electrophoresis identification, selects sequencing correctly clone
Carry out a large amount of plasmid extractions.Then it is linearized with restriction enzyme NruI, ethanol precipitation recycles plasmid, 95 DEG C of heating
15min degerming, after room temperature is cooling, -20 DEG C are saved backup.
Secondly, above-mentioned recombinant expression carrier transfected Chinese hamster gonad cell (CHO-S cell) and express express target protein:
Using liposome method (Freestyle MAX, invitrogen), by plasmid pCHO 1.0-GLP-1- after linearisation
Fc-6 distinguishes transfected CHO-S cells, detects GLP-1-Fc expressing fusion protein situation.Plasmid transfection is according to FreestyleTMMAX
Transfection reagent operation instructions, CHO-S cell transfecting the previous day is according to 0.5 × 106/ ml passage, the transfection same day count, and adjustment is thin
Born of the same parents' density is 1 × 106/ ml, 37 DEG C of cultures.Then Transfection solution is prepared, by 50ug linearization plasmid pCHO 1.0-GLP-1-
OptiPRO is added in Fc-6TMSFM culture medium is to 1.5ml, 50ul FreestyleTM1.45ml is added in MAX transfection reagent
OptiPROTMTransfection reagent mixed liquor is slowly added into DNA mixed liquor by SFM culture medium after mixing respectively, mixes room temperature
It stands after ten minutes, transfection cocktail is slowly added dropwise in the CHO-S cell of 30ml.8%CO2, 37 DEG C, 130rpm culture.
After transfecting 48h, the Puromycin and MTX of various concentration pressurize, and pressurize through 2 wheels, and cell viability restores to 90%, freezes 4；
And take part cell with 0.3 × 106/ ml is inoculated with 30ml, and feed-batch culture collects supernatant.Through testing goal expressing quantity
4897.7ug/ml。
In another embodiment, by the polynucleotides of encoding amino acid sequence signal peptide as shown in SEQ ID NO:6 with
The polynucleotides connection rear clone of coding FGF21 cell factor enters FreedompCHO1.0, and transfected HEK 293 is expressed.
It is compared the result shows that signal peptide of the present invention carries signal peptide with FGF21, may make the expression quantity of FGF21 cell factor certainly
353ug/ml is improved to 2563ug/ml, improves 7.26 times.This signal peptide also significantly increases the expression quantity of cell factor.
In another embodiment, by the polynucleotides of encoding amino acid sequence signal peptide as shown in SEQ ID NO:6 with
The polynucleotides connection of coding PD-1 heavy chain of antibody is cloned into pCDNA3.3 carrier, and light chain vector and signal peptide are constant, two loads
Body cotransfection CHO-K1 cell express express target protein.The result shows that the heavy chain signal peptide that signal peptide of the present invention and former project carry
The signal peptide of human albumin is compared, and the expression quantity of PD-1 antibody may make to be increased to 6.58g/L, expression quantity by 0.52g/L
Improve 12.65 times.This signal peptide can also be used for improving the expression quantity of other antibody such as anti-CD 20 antibodies.
The present invention provides a kind of for guiding the artificial synthesized letter of foreign protein secreting, expressing in mammalian cells
Number peptide, signal peptide of the present invention is compared with other signal peptide for antibody, the measurement result of exogenous protein expression amount shows this hair
Bright signal peptide is expressed especially suitable for the heterologous protein secretion of mammalian cell, dynamic in lactation compared with other signal peptides
Exogenous protein expression amount in object cell is substantially improved, and expression improves 6 times or more, significantly improves external source egg
White secreting, expressing amount, application is conducive to screen high expression monoclonal cell strain, and can reduce production cost, has extensive
Prospects for commercial application.
Detailed description of the invention
Fig. 1 is Freedom pCHO1.0 expression vector map.
Fig. 2 is pCHO1.0-GLP-1-Fc plasmid double digestion qualification figure；
M,thermo marker；
1, pCHO1.0-SP1-GLP-1-Fc double digestion；
2, pCHO1.0-SP2-GLP-1-Fc double digestion；
3, pCHO1.0-SP3-GLP-1-Fc double digestion；
4, pCHO1.0-SP4-GLP-1-Fc double digestion；
5, pCHO1.0-SP5-GLP-1-Fc double digestion；
6, pCHO1.0-SP6-GLP-1-Fc double digestion.
Fig. 3 is the standard curve of Fortebio measurement unlike signal peptide expression GLP-1-Fc albumen；
Fig. 4 is the SDS-PAGE electrophoresis of the GLP-1-Fc of signal peptide and the guidance expression of other signals peptide of the invention；
M, low molecular weight protein Marker；
1, the GLP-1-Fc of signalase 11 inducing expression；
2, the GLP-1-Fc of 2 inducing expression of signal peptide；
3, the GLP-1-Fc of 3 inducing expression of signal peptide；
4, the GLP-1-Fc of 4 inducing expression of signal peptide；
5, the GLP-1-Fc of 5 inducing expression of signal peptide；
6, the GLP-1-Fc of signal peptide inducing expression of the present invention；
Fig. 5 is the purity detecting result figure of the GLP-1-Fc albumen of signal peptide expression of the present invention；GLP-1-Fc albumen goes out
Peak time is 9.667min, and the peak 12min is solvent peak.
Specific embodiment
Following embodiments technical solution that the present invention will be described in detail, but protection scope of the present invention is not limited.
Utilize the method for signal peptide of the invention secreting, expressing protein in mammalian cells are as follows: will the coding present invention
The polynucleotides of signal peptide connect rear clone with the polynucleotides of coding expression protein and enter mammalian cell expression vector, so
Afterwards by express express target protein after the recombinant mammalian cells expression vector transfection mammalian cell.
Polynucleotides of the invention can be prepared by conventional synthetic method.
Expression vector cited by the embodiment of the present invention is Freedom pCHO1.0 (being purchased from Invitrogen).
Embodiment 1
(1) synthesis of signal peptide and the synthesis of GLP-1-Fc polynucleotide sequence
Signal peptide sequence is short, is generally made of 51~60 nucleotide, the multicore of signal peptide and GLP-1-Fc fusion protein
Nucleotide sequence synthesizes jointly.II restriction enzyme site of restriction enzyme A vr, initiation codon ATG, Kozak sequence are added before signal peptide
Column；Add terminator codon TGATGA and I restriction enzyme site of restriction enzyme BstZ17 in the fusion protein sequence end GLP-1-Fc；
All polynucleotide sequences have the synthesis of Shanghai bioengineering Co., Ltd.
The present invention devises artificial synthesized novel signal peptide 6, at the same the signalase 11-5 that provides of application article and patent into
Row comparison.
The amino acid sequence of signalase 11-6 is as follows:
Signalase 11 (SEQ ID NO:1): MKWVTFISLLFLFSSAYS-human albumin；
Signal peptide 2 (SEQ ID NO:2): MTRLTVLALLAGLLASSRA-Azurocidinpreproprotein；
Signal peptide 3 (SEQ ID NO:3): MDVPAEFLGLLLLWLSGVRC-CN102659928A；
Signal peptide 4 (SEQ ID NO:4): MKWVKVLFALICIAVAHS-CN106478773A；
Signal peptide 5 (SEQ ID NO:5): MEFGLSWVFLVALFRGVQC-H7；
Signal peptide 6 (SEQ ID NO:6): the MGKLIFWLVFWLTIFWLGSA- present invention.
The nucleotide sequence of signalase 11-6 is as follows:
Signalase 11 (SEQ ID NO:7):
5'-ATGAAGTGGGTCACCTTCATCTCCCTTCTCTTTTTGTTCAGTTCCGCTTACAGC-3'；
Signal peptide 2 (SEQ ID NO:8):
5’
-ATGACCAGACTGACCGTGCTGGCCCTGCTGGCGGGCCTGCTGGCTTCTTCTAGAGCC-3'；
Signal peptide 3 (SEQ ID NO:9):
5’
-ATGGATGTTCCTGCCGAATTTCTTGGATTGTTGTTGTTGTGGCTCTCCGGAGTGCGTTGC-3'；
Signal peptide 4 (SEQ ID NO:10):
5’
-ATGAAGTGGGTGAAGGTGCTGTTCGCCCTGATCTGCATCGCCGTGGCCCATAGC-3'；
Signal peptide 5 (SEQ ID NO:11):
5’
-ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTTAGAGGTGTCCAGTGT-3'；
Signal peptide 6 (SEQ ID NO:12):
5’
-ATGGGCAAACTCATATTCTGGCTTGTGTTTTGGCTGACTATTTTTTGGCTTGGATCAGCT-3'；
(2) building of pCHO1.0 expression vector is recombinated
Synthesized by Shanghai bioengineering Co., Ltd and constructed 6 kinds of signal peptide difference plasmid puc 57-GLP-1-Fc-1,
puc57-GLP-1-Fc-2、puc57-GLP-1-Fc-3、puc57-GLP-1-Fc-4、puc57-GLP-1-Fc-5、puc57-
After extracting plasmids in a small amount, with restriction enzyme A vr II, I double digestion of BstZ17, it is solidifying then to carry out agarose by GLP-1-Fc-6
Gel electrophoresis, glue recycle target fragment.By target fragment and the equally eukaryon through restriction enzyme A vr II, I double digestion of BstZ17
Expression vector pCHO1.0 connection constructs recombinant eukaryon expression vector pCHO 1.0-GLP-1-Fc-1, pCHO 1.0-GLP-1-
Fc-2、pCHO1.0-GLP-1-Fc-3、pCHO 1.0-GLP-1-Fc-4、pCHO 1.0-GLP-1-Fc-5、pCHO 1.0-GLP-
1-Fc-6 converts E. coli DH5 α, is incubated overnight rear picking positive colony and carries out mini-scale plasmid extraction, restricted
Agarose gel electrophoresis is identified after restriction endonuclease Avr II, I double digestion of BstZ17, and double digestion map is as shown in Fig. 2, will the identification positive
Clone serve the sequencing of marine growth Engineering Co., Ltd, correctly clone carries out a large amount of plasmid extractions for selection sequencing.Then with limit
Property restriction endonuclease NruI linearisation processed, ethanol precipitation recycle plasmid, and 95 DEG C of heating 15min degermings, after room temperature is cooling, -20 DEG C are protected
It deposits spare.
(3) recombinant expression carrier transfected Chinese hamster gonad cell (CHO-S cell) and express express target protein
Using liposome method (Freestyle MAX, invitrogen), by plasmid pCHO 1.0-GLP-1- after linearisation
Fc-1、pCHO1.0-GLP-1-Fc-2、pCHO 1.0-GLP-1-Fc-3、pCHO 1.0-GLP-1-Fc-4、pCHO 1.0-GLP-
1-Fc-5, pCHO 1.0-GLP-1-Fc-6 distinguish transfected CHO-S cells, detect GLP-1-Fc expressing fusion protein situation.Plasmid
Transfection is according to FreestyleTMMAX transfection reagent operation instructions, CHO-S cell transfecting the previous day is according to 0.5 × 106/ ml is passed
Generation, the transfection same day count, and adjustment cell density is 1 × 106/ ml, 37 DEG C of cultures.Then Transfection solution is prepared, 50ug is linear
Change plasmid pCHO1.0-GLP-1-Fc-1, pCHO1.0-GLP-1-Fc-2, pCHO1.0-GLP-1-Fc-3, pCHO1.0-GLP-1-
Fc-4, pCHO1.0-GLP-1-Fc-5, pCHO1.0-GLP-1-Fc-6 are separately added into OptiPROTMSFM culture medium to 1.5ml,
50ul FreestyleTM1.45ml OptiPRO is added in MAX transfection reagentTMAfter mixing respectively, transfection is tried for SFM culture medium
Agent mixed liquor is slowly added into DNA mixed liquor, and mixing is stored at room temperature after ten minutes, and transfection cocktail is slowly added dropwise into 30ml
CHO-S cell in.8%CO2, 37 DEG C, 130rpm culture.After transfecting 48h, the Puromycin and MTX of various concentration pressurize,
Through 2 wheel pressurizations, cell viability restores to 90%, freezes 4；And take part cell with 0.3 × 106/ ml is inoculated with 30ml, feed supplement training
It supports, collects supernatant.
(4) Fortebio detects the expression of destination protein in culture supernatant
After GLP-1-Fc standard items are diluted to 500ug/ml with PBST, 8 concentration of doubling dilution do standard curve, use
PBST4 times dilute plasmid pCHO 1.0-GLP-1-Fc-1, pCHO 1.0-GLP-1-Fc-2, pCHO 1.0-GLP-1-Fc-3,
PCHO1.0-GLP-1-Fc-4, pCHO 1.0-GLP-1-Fc-5, pCHO 1.0-GLP-1-Fc-6F distinguish transfected CHO-S cells
Culture supernatant, select ProteinA biosensor, detect supernatant in destination protein concentration, determine destination protein expression quantity,
As a result as shown in Fig. 3, table 1.
Influence of the 1 unlike signal peptide of table to GLP-1-Fc fusion protein secreting, expressing
From table 1 it follows that signal peptide of the present invention can increase substantially the secreting, expressing of GLP-1-Fc fusion protein
Amount, so that heterologous protein secretion expression quantity improves 6.23 times compared with original signal peptide human albumin.
(5) SDS-PAGE testing goal molecular weight of albumen and expression quantity
Whether the supernatant destination protein that SDS-PAGE reduction electrophoresis detection embodiment 3 is collected expresses.According to Chinese Pharmacopoeia
The method of three annex carries out electrophoresis, and electrophoretogram gray scale scanning identifies its molecular weight and expression quantity, and electrophoresis result is as shown in Figure 4.
(6) destination protein in purifying cells culture supernatant
Using ProteinA affinity chromatography.ProteinA affinity column is prepared first, after balancing pillar with PBS, will be centrifuged
And the cells and supernatant of 0.4um membrane filtration crosses column, is then washed till OD value close to zero, with 50mmol/L pH7.5 with PBS
Glycine-HCI solution elution, collect the eluent of peak region, it is spare after dialysing.
(7) purity of HPLC method measurement GLP-1-Fc
HPLC method measures the purity of GLP-1-Fc, and chromatographic column is Tskgel G3000SWXL, and mobile phase is 0.1M Na2SO4-
0.1M Na2HPO4(pH6.7), loading 10ul, 280nm detection.GLP-1-Fc is in 9.667min appearance, purity of protein
99.17%, HPLC test map are as shown in Figure 5.
Sequence table
<110>Lunan Pharmaceutical Group Co., Ltd.
<120>a kind of artificial synthesized signal peptide
<130> 2018
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> PRT
<213> Human
<400> 1
Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala
1 5 10 15
Tyr Ser
<210> 2
<211> 19
<212> PRT
<213> Azurocidin
<400> 2
Met Thr Arg Leu Thr Val Leu Ala Leu Leu Ala Gly Leu Leu Ala Ser
1 5 10 15
Ser Arg Ala
<210> 3
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Met Asp Val Pro Ala Glu Phe Leu Gly Leu Leu Leu Leu Trp Leu Ser
1 5 10 15
Gly Val Arg Cys
20
<210> 4
<211> 18
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Lys Trp Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala
1 5 10 15
His Ser
<210> 5
<211> 19
<212> PRT
<213> Human
<400> 5
Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Phe Arg Gly
1 5 10 15
Val Gln Cys
<210> 6
<211> 20
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Met Gly Lys Leu Ile Phe Trp Leu Val Phe Trp Leu Thr Ile Phe Trp
1 5 10 15
Leu Gly Ser Ala
20
<210> 7
<211> 54
<212> DNA
<213> human
<400> 7
atgaagtggg tcaccttcat ctcccttctc tttttgttca gttccgctta cagc 54
<210> 8
<211> 57
<212> DNA
<213> Azurocidin
<400> 8
atgaccagac tgaccgtgct ggccctgctg gcgggcctgc tggcttcttc tagagcc 57
<210> 9
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
atggatgttc ctgccgaatt tcttggattg ttgttgttgt ggctctccgg agtgcgttgc 60
<210> 10
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
atgaagtggg tgaaggtgct gttcgccctg atctgcatcg ccgtggccca tagc 54
<210> 11
<211> 57
<212> DNA
<213> Human
<400> 11
atggagtttg ggctgagctg ggttttcctc gttgctcttt ttagaggtgt ccagtgt 57
<210> 12
<211> 60
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
atgggcaaac tcatattctg gcttgtgttt tggctgacta ttttttggct tggatcagct 60
<210> 13
<211> 825
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
cacggggaag gcacttttac tagcgacgtt agttcatatc tcgaagaaca agccgcaaaa 60
gaatttatcg catggctggt gaagggaggt ggtggaggtg gtggatcagg gggtggcggt 120
agcggtggtg ggggcagcgc agaaagcaag tacggaccac cctgtccccc atgccccgcc 180
cccgaagctg ccggtggccc tagtgtgttt ctgttccccc ctaagcctaa ggacactctg 240
atgatttcac gaacacctga agtcacatgt gtcgtcgtcg atgtctccca agaagatcca 300
gaggtacagt tcaactggta tgtggatggg gtcgaggtcc acaacgccaa aactaaacct 360
cgggaagagc agtttaatag tacatataga gtggtcagtg tccttactgt gcttcatcag 420
gattggttga atgggaaaga gtataaatgt aaagtgagca acaaggggct gccttcttct 480
attgagaaga ctatttcaaa agctaaagga caaccaagag agccccaagt atacacactt 540
cccccatcac aggaggagat gacaaaaaac caggtatctc tcacatgcct tgttaagggg 600
ttctatcctt ccgacatcgc agtagaatgg gaaagtaatg ggcaaccaga aaataactat 660
aaaaccacac ctcctgtgct ggatagtgac gggtcctttt ttttgtactc tagactgaca 720
gtggacaaat cccgctggca ggaaggaaat gttttttcct gtagcgtgat gcacgaggca 780
cttcacaacc actacaccca aaaaagcctt tccctgtctc tgggt 825
Claims (10)
1. a kind of artificial synthesized signal peptide, amino acid sequence is as shown in SEQ ID NO:6.
2. a kind of external source GLP-1-Fc fusion protein, FGF21 cell factor, PD-1 antibody, anti-CD 20 antibodies secreting, expressing side
Method, which is characterized in that use the signal peptide as shown in SEQ ID NO:6.
3. a kind of polynucleotides, signal peptide described in the polynucleotide encoding claim 1.
4. polynucleotides according to claim 3, which is characterized in that the sequence of the polynucleotides such as SEQ ID:12
It is shown.
5. a kind of mammalian cell expression vector, the expression vector contains the polynucleotides of claim 3 or 4 and coding
The polynucleotides of the protein of mammal source.
6. mammalian cell expression vector according to claim 5, which is characterized in that the multicore of claim 3 or 4
Thuja acid is immediately in the polynucleotides front end of the encoding mammalian derived protein.
7. mammalian expression vector according to claim 6, which is characterized in that the promoter of the expression vector is EF-1
α promoter, hCMV promoter, the hybrid promoter of SV40 promoter or EF-1 α and hCMV promoter.
8. a kind of mammalian host cell, the host cell is transfected by any expression vector of claim 5-7, institute
Stating mammalian cell is CHO, BHK, HEK293 or SP2/0.
9. signal peptide described in claim 1 guides external source fusion protein, cell factor or antibody in mammalian host cell
The purposes of secreting, expressing.
10. signal peptide described in claim 1 guides external source GLP-1-Fc fusion protein, FGF21 in mammalian host cell
The purposes of cell factor, PD-1 antibody, anti-CD 20 antibodies secreting, expressing.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810432932.1A CN110452288A (en) | 2018-05-08 | 2018-05-08 | A kind of artificial synthesized signal peptide |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810432932.1A CN110452288A (en) | 2018-05-08 | 2018-05-08 | A kind of artificial synthesized signal peptide |
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Family
ID=68480494
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| CN201810432932.1A Pending CN110452288A (en) | 2018-05-08 | 2018-05-08 | A kind of artificial synthesized signal peptide |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112028972A (en) * | 2020-08-27 | 2020-12-04 | 北京百普赛斯生物科技股份有限公司 | Signal peptide for improving secretion expression of recombinant protein of mammalian cell and application thereof |
| CN113402589A (en) * | 2021-06-18 | 2021-09-17 | 苏州工业园区唯可达生物科技有限公司 | Signal peptide for improving antibody yield |
| CN114539357A (en) * | 2020-11-27 | 2022-05-27 | 广州汉腾生物科技有限公司 | Application of signal peptide in expression of GLP-1 fusion protein |
-
2018
- 2018-05-08 CN CN201810432932.1A patent/CN110452288A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112028972A (en) * | 2020-08-27 | 2020-12-04 | 北京百普赛斯生物科技股份有限公司 | Signal peptide for improving secretion expression of recombinant protein of mammalian cell and application thereof |
| CN114539357A (en) * | 2020-11-27 | 2022-05-27 | 广州汉腾生物科技有限公司 | Application of signal peptide in expression of GLP-1 fusion protein |
| WO2022110499A1 (en) * | 2020-11-27 | 2022-06-02 | 广州汉腾生物科技有限公司 | Application of signal peptide in expression of glp-1 fusion protein |
| CN113402589A (en) * | 2021-06-18 | 2021-09-17 | 苏州工业园区唯可达生物科技有限公司 | Signal peptide for improving antibody yield |
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