CN1288059A - Yeast expression for secretory fusion protein - Google Patents
Yeast expression for secretory fusion protein Download PDFInfo
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- CN1288059A CN1288059A CN 99116862 CN99116862A CN1288059A CN 1288059 A CN1288059 A CN 1288059A CN 99116862 CN99116862 CN 99116862 CN 99116862 A CN99116862 A CN 99116862A CN 1288059 A CN1288059 A CN 1288059A
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Abstract
The present invention relates to a method for expressing the secretory granulocyte-macrophage colony stimulating factor (GM-CSF) and fusion protein of interleukin 3 (IL-3) in yeast, the engineering bacteria for preparing said fusion protein, and the resultant fusion protein MX. Said fusion protein MX gene is very stable and can be controlled to expression. Its resultant has natural amino acid protein sequence without replacement mutation.
Description
The present invention relates to bioengineering field, relate more specifically to the method for the rHuGM-CSF (GM-CSF) of expression-secretion in yeast and the fusion rotein of interleukin (IL-3) (abbreviating " MX fusion rotein " as), also relate to the engineering bacteria of producing this fusion rotein and the fusion rotein MX that is produced.
RHuGM-CSF (GM-CSF) and interleukin (IL-3) are two kinds of cytokines that play an important role in human body.Two kinds of cytokines are merged the fusion rotein that forms (abbreviating " MX fusion rotein " as), can have both advantages.At present, existing technological method with yeast expression MX fusion rotein, but this method has the shortcoming of following several respects:
(1) utilize the DNA plasmid to change the MX antigen-4 fusion protein gene in the prior art, but the antigen-4 fusion protein gene of introducing is not integrated in people's zymic chromatin.Therefore, the gene of MX fusion rotein is lost easily in growth expression process.
(2) the MX Expression of Fusion Protein can't induction regulating controlling.At present, the report that does not still have abduction delivering MX fusion rotein.
(3) original MX fusion protein product can be by the excess glycosylation.Thereby can only on original gene, carry out the amino acid mutation displacement to avoid by the excess glycosylation.So that final product albumen has several (6) replacement amino acids to be different from natural product.
Thereby this area presses for to provide and has native sequences and not by glycosylated MX fusion rotein of excess and production method thereof.
Purpose of the present invention overcomes above-mentioned shortcoming of the prior art exactly, provide a kind of and express the method for production MX fusion rotein by yeast stability and high efficiency ground, and the GM-CSF in this MX fusion rotein and IL-3 can have the aminoacid sequence of natural mature protein.
In a first aspect of the present invention, the method of the fusion rotein MX of a kind of rHuGM-CSF of expression-secretion and interleukin is provided, this method comprises: (a) encoding sequence of rHuGM-CSF and the encoding sequence of interleukin are merged, form the polynucleotide sequence of encoding fusion protein MX; (b) polynucleotide sequence with encoding fusion protein MX in the step (a) is inserted into expression vector, forms the expression vector that contains fusion rotein MX encoding sequence; (c) change the expression vector that contains fusion rotein MX encoding sequence in the step (b) over to yeast cell; (d) filter out the MX encoding sequence and be integrated into genomic transformed yeast cells; (e) under the condition that is fit to coding MX fusion rotein, transformed yeast cells in the culturing step (d); (f) isolate the MX fusion rotein.Preferably, this fusion rotein MX has the aminoacid sequence shown in the SEQ ID NO:2.
In a second aspect of the present invention, provide with the rHuGM-CSF of method for preparing and the fusion rotein MX of interleukin.Preferably, the rHuGM-CSF of described rHuGM-CSF and interleukin derived from human and interleukin, best, the aminoacid sequence that has sophisticated natural human rHuGM-CSF and interleukin.
Aspect the 3rd of the present invention, a kind of yeast cell is provided, be integrated with the polynucleotide of the fusion rotein MX of the rHuGM-CSF of expression-secretion and interleukin in this zymic genome, and the fusion rotein of this nucleotide coding aminoacid sequence shown in SEQ ID NO:2.Preferably, this yeast cell is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) or pichia pastoris phaff (Pichiapastoria).More preferably, this cell is to be preserved in Chinese typical culture collection center on August 6th, 1999, and preserving number is pichia pastoris phaff (Pichia pastoria) MX of CCTCC No.:M99009.
In the present invention, term " fusion rotein MX " and " MX fusion rotein " are used interchangeably, and all refer to merge formed polypeptide by rHuGM-CSF (GM-CSF) and two kinds of albumen of interleukin (IL-3).Described rHuGM-CSF and interleukin can be derived from human, also can be derived from inhuman Mammals, but be preferably the rHuGM-CSF and the interleukin of derived from human.In addition, GM-CSF and IL-3 can have natural wild-type GM-CSF and IL-3, also can be the GM-CSF of mutant and IL-3 (as long as the GM-CSF of sudden change and IL-3 also have all the activity with the part wild-type protein).GM-CSF in the MX fusion rotein and IL-3 can have signal peptide sequence, but preferably do not have the original signal peptide and only have the aminoacid sequence of maturation protein.The example of a kind of fusion rotein MX has the aminoacid sequence shown in the SEQ ID NO:2.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is a yeast cell.More preferably, this host cell is a pichia.Be pichia pastoris phaff best.
In the present invention, the gene of encoding fusion protein MX can obtain by methods such as PCR method or direct labor synthesize.For example, first RT-PCR method obtains the encoding sequence of GM-CSF and IL-3 respectively, then they is merged and forms the gene of encoding fusion protein MX.
After having obtained fusion rotein MX gene, can be inserted in the suitable carriers, so that transform and or identify.Be applicable to that carrier of the present invention comprises various carrier known in the art, as commercially available carrier.For example select commercially available carrier for use, the nucleotide sequence with code book invention polypeptide is inserted into suitable site then, and the integrated expression vector of reorganization can take place with the yeast genes group in formation.Integrating vector is meant and can the DNA reorganization takes place with the genome of host cell, thereby exogenous origin gene integrator is gone into carrier in the host cell gene group.The example of suitable integrated expression vector comprises (but being not limited to): plasmid pPIC9, plasmid p α ADH2 etc.
There are numerous known ordinary methods to change host cell over to, comprising (but being not limited to): electroporation, lyticase method (lyticase) and chemical method with containing the expression of exogenous gene carrier.
After changing expression vector over to host cell, can be screened according to corresponding marker gene, for example screen according on the MD substratum, growing.
Filter out conversion and host cell that in genome, be integrated with fusion rotein MX gene, just can under the condition that is fit to fusion rotein MX expression, cultivate this transformed host cells.Because fusion rotein MX of the present invention is a secretion property, so fusion rotein MX is secreted in nutrient solution.
After having obtained to contain the nutrient solution of fusion rotein MX, can carry out separation and purification with ordinary method known in the art, thereby obtain the fusion rotein MX of purifying.Suitable separation purification method comprises: ion exchange chromatography, sieve chromatography, precipitation of protein, affinity chromatography, HPLC etc.
Production MX fusion rotein method of the present invention and engineering bacteria have the following advantages:
(1) the MX antigen-4 fusion protein gene is integrated in people's yeast chromatin, thereby can grow steadily in the long term and express unlikely being lost.
(2) genetic expression of MX fusion rotein can be by induction regulating controlling.In an example of the present invention, come induction regulating controlling with methyl alcohol.
(3) original MX fusion protein product can be by the excess glycosylation.Thereby the gene that uses the sudden change of former beginning and end is as expression template.The product that gives expression to is the natural amino acid protein sequence, and no amino acid sites is replaced.
In brief, MX antigen-4 fusion protein gene of the present invention is very stable, controllable express, and product is the natural amino acid protein sequence, no replacement mutation.
The present invention also provides the pharmaceutical composition that contains fusion rotein MX of the present invention, and it contains fusion rotein MX and the pharmaceutically acceptable carrier or the vehicle for the treatment of significant quantity.Suitable carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Pharmaceutical preparation should be complementary with administering mode.Fusion rotein MX of the present invention can be made into the injection form, for example is prepared by ordinary method with the physiological saline or the aqueous solution that contains glucose and other assistant agents.Pharmaceutical composition such as tablet and capsule can be prepared by ordinary method.Pharmaceutical composition such as injection, solution, tablet and capsule should be made under aseptic condition.The dosage of activeconstituents is the treatment significant quantity, for example every day about 1 microgram/kg body weight-Yue 5 mg/kg body weight.In addition, fusion rotein MX of the present invention also can use with the other treatment agent.
When fusion rotein MX of the present invention is used as medicine, this polypeptide of treatment effective dose can be applied to Mammals, wherein should treat effective dose usually at least about 10 micrograms/kg body weight, and in most of the cases be no more than about 8 mg/kg body weight, preferably this dosage is about 10 micrograms/kg body weight-Yue 1 mg/kg body weight.Certainly, concrete dosage also should be considered factors such as route of administration, patient health situation, and these all are that skilled practitioners can be determined within skill.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to people such as normal condition such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
With RT-PCR method clone GM-CSF and IL-3 CDNA '
With people's normal liver cell strain ATCC CCL13 grown cultures in the DMEM nutrient solution that contains 10% calf serum, in carbon dioxide cell incubator 37 ℃, 5% CO
2Cultivate.Results 1 * 10
8Individual CCL13 cell.The RNA extraction column of producing with Qiagen company carries out the RNA extracting to the CCL13 cell of gathering in the crops.The RNA that obtains is as the template of reverse transcribing (RT) reaction.With random primer (Random Primer) and MMLV reverse transcriptase the RNA that extracts is carried out reverse transcribing.The product that obtains is as the template of next step archaeal dna polymerase chain reaction (PCR).
With following two pairs of primers, MX-1 and MX-2, MX-3 and MX-4, MX-1:5 ' GCTCTA GAGGAGGATGTGGCTGCAGA 3 ' (SEQ ID NO:3) MX-2:5 ' GCG GATCCGCCGCCACCCCCAGATCCACCGCCACCCTCCTGGACTGGCTCCCAG 3 ' (SEQ ID NO:4) MX-3:5 ' CGGATCCGCTCCCATGACCCAGACAAC 3 ' (SEQ ID NO:5) MX-4:5 ' CGGATCCGAACGAGCTGGACGTTGGAC 3 ' (SEQ ID NO:6) with above-mentioned reverse product of transcribing as template, carry out archaeal dna polymerase chain reaction (PCR) respectively, to amplify the encoding sequence of human GM-CSF and IL-3.
Embodiment 2
Connect GM-CSF cDNA and IL-3 cDNA and form the MX fusion gene
PCR product (being the encoding sequence of GM-CSF) to primer MX-1 and MX-2 digests with restriction endonuclease Xba I and BamH I.PCR product (being the encoding sequence of IL-3) to primer MX-3 and MX-4 digests with restriction endonuclease BamH I.Work plasmid pBluescript digests with Xba I and BamH I.
Three products of digestion are separately connected at 16 ℃ with T4 dna ligase (ligase).Connect the product transformed into escherichia coli.Then, extracting DNA plasmid also carries out restriction analysis, determines wherein to have formed GM-CSF and the IL-3 DNA that links into an integrated entity, and is called the PMX fusion gene.
Embodiment 3
The MX fusion gene that is used for yeast expression with PCR method clone
As template, carry out the PCR reaction to clone the MX fusion gene that is used for yeast expression with the PMX fusion gene of above-mentioned identification with following primer 1 and 2.
Primer 1:
5′GCTCGAGAAAAGAGCACCCGCCCGCTCGCCC?3′(SEQ?ID?NO：7)
Primer 2:
5′CGGATCCGAACGAGCTGGACGTTGGAC?3′(SEQ?ID?NO：8)
The product of PCR so in the work plasmid Bluescript that inserts with Xho I and the digestion of BamH I, transforms through intestinal bacteria with restriction endonuclease Xho I and the digestion of BamH I, the DNA plasmid extraction, and restriction endonuclease digestion is analyzed.Contain the plasmid of the correct fusion rotein MX gene that is used for yeast expression, be named as Bluescript.mx.With Xho I and Not I digestion Bluescript.mx, MX fusion gene with gained inserts yeast plasmid pPIC9 (Gene 105:205 then, 1991) (many specific cerevisiae dna orders are arranged on this plasmid, comprise secretion signal, select gene and the sequence that is recombined into the yeast chromosomal site.) Xho I and Not I site, form plasmid P-MX.P-MX is carried out DNA sequence measure, confirm that the MX fusion gene has correct nucleotide sequence, (does not contain signal peptide sequence) shown in SEQID NO:1.
Embodiment 4
Change the MX fusion gene over to pichia pastoris phaff (Pichia pastoria) with electric shocking method
In the YPD nutrient solution, 28 ℃ are shaken grow overnight with pichia pastoris phaff (Pichia pastoria) GS115 inoculation.Next day, yeast concn is between O.D.6002-6.Yeast and the postdigestive plasmid P-MX of restriction endonuclease Sal I are mixed, place the electric shock instrument to shock by electricity then with 7500 volts/cm 5-10 millisecond.Yeast after the electric shock is applied to MD and selects to put 28 ℃ and cultivate on the substratum.Selected clone after 3-5 days.Single clonal growth in the MD substratum, was cultivated 36 hours in 28 ℃.Selected clone carries out dna sequencing again, reaffirms that the MX fusion gene is correct.
A strain positive colony that filters out is preserved in Chinese typical culture collection center on August 6th, 1999, and preserving number is CCTCC No.:M99009, called after " pichia pastoris phaff (Pichia pastoria) MX ".
Embodiment 5
MX fusion gene product is expressed
Select the yeast clone after the conversion of embodiment 4, be inoculated in the 25ml BMGY nutrient solution.With 28-30 ℃, 250-300rpm speed wave and culture spends the night in the 250ml Erlenmeyer flask.Next day is yeast centrifugation, and suspending again with the BMMY nutrient solution, to make concentration be O.D.600=1.0 to yeast.Place triangular flask to continue to cultivate the yeast juice that suspends again at said temperature and the speed of shaking.The methyl alcohol that added 100% concentration every 24 hours is induced, and making final concn is 0.5%.Respectively got 5ml yeast culture liquid at 0,24,48,72,96 hour as sample.The sample of collecting gives 15, and 000g is centrifugal, collects supernatant, freezes in-20 ℃ to use for analyzing.
Embodiment 6
The analysis of MX fusion gene expression product
The poly-propionic acid amide gel of SDS-of poly-propionic acid amide gel electrophoresis (SDS-PAGE) preparation 12.5% of SDS-, the supernatant sample of every 70 each time of microlitre of Kong Shangdian.Dye with Coomassie brilliant blue behind the electrophoresis.At molecular weight about 50,000 visible disperse protein bands.The amount of this protein band increased with the abduction delivering time, and its molecular weight equals GM-CSF and IL-3 molecular weight sum, did not therefore have excess saccharification phenomenon.Not with there not being this protein band in the yeast supernatant liquor of MX fusion gene.
The 3rd day supernatant sample 25 microlitres of western blot test (Western blot) abduction delivering are splined on 12.5% the poly-propionic acid amide gel of SDS-.Be transferred on the pvdf membrane behind the electrophoresis.Pvdf membrane after the transfer is used anti-human GM-CSF and anti-people IL-3 monoclonal antibody (R﹠amp respectively; D System) reacts.Use the sheep anti-mouse igg second antibody (Sigma) of alkali phosphatase enzyme mark to react then.Behind the substrate staining, in the expression supernatant of MX fusion gene transformed yeast visible one respectively with the band of anti-human GM-CSF, anti-people IL-3 antibody response.This band is the disperse shape, molecular weight about 50,000.In the yeast expression supernatant of contrast, there is not band with above-mentioned two antibody responses.
It is to separate a cell strain of setting up from the leukemia human blood that external TF-1 cell thing heteroplasia lunge swashs test TF-1 cell.This cell strain has special growth dependency for GM-CSF and IL-3.Promptly only in the nutrient solution that contains GM-CSF and IL-3, the TF-1 cell could divide amplification.Utilize the growth dependency of this cell strain, can carry out the specific function test GM-CSF and IL-3.
In 96 porocyte culture plates, every hole adds 50 microlitre basic culture solutions (RPMI-1640 contains 2mM L-glutamine (L-glutamine), the blue or green laminin of 100 units/ml, 100 micrograms/ml chain laminin and 10% calf serum).Add 25 microlitre specimen in first round, get 25 microlitre to the second rounds behind the mixing, again serial dilution like this.It is basic culture solution only that last two rounds keep, as negative control.Each hole adds 50 microlitre TF-1 cell suspension C cell countings 2 * 10
5/ m is in basic culture solution), 37 ℃, 5%CO
2Cultivated 4-5 days.Observe counting.With aforesaid method the MX transformed yeast being expressed supernatant tests.The supernatant of expressing the 3rd day still has differential stimulus TF-1 cell growth effect after dilution in continuous 33 times 1: 3 is 333 times of dilutions.There is not TF-1 cytositimulation growth activity and respectively contrast the yeast expression supernatant liquor.
Comprehensive above-mentioned experimental result shows that the yeast that the MX fusion gene transforms can be by first ferment abduction delivering, the albumen of a part amount about 50,000.Its molecular weight equals GM-CSF and IL-3 molecular weight sum, thereby does not have excess saccharification phenomenon.This marking protein is secreted in the nutrient solution thereby is easy to separation and purification.This yeast expression MX output height can be by the Coomassie brilliant blue recognition expression product that dyes with the original expression supernatant of 70 microlitres.Expression product can be by the antibody recognition of anti-human GM-CSF and anti-people IL-3.Show that this albumen has the antigenicity of human GM-CSF and people IL-3 simultaneously.Functional examination with external GM-CSF and IL-3 dependent cell strain TF-1 shows that this expression product has high degree of specificity TF-1 growth stimulating activity.The expression supernatant still has the special growth stimulation to the TF-1 cell after being 333 times of dilutions through dilution in continuous 33 times 1: 3.The yeast supernatant of all contrasts is not all seen the proteic characteristic of above-mentioned MX.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
(1) general information:
(ⅰ) applicant: Zhao-an Medical Science ﹠ Tech. Co., Ltd., Shanghai
(ⅱ) denomination of invention: the yeast expression of secretory fusion protein
(ⅲ) sequence number: information (ⅰ) sequence signature of (2) SEQ ID NO.1:
(A) length: 816bp
(B) type: Nucleotide
(C) chain: two strands
( D ) ： ( ⅱ ) ： ( ⅸ ) ：SEQ ID NO.1：GCA CCC GCC CGC TCG CCC AGC CCC AGC ACG CAG CCC TGG GAG CAT GTG AAT 51 A P A R S P S P S T Q P W E H V NGCC ATC CAG GAG GCC CGG CGT CTC CTG AAC CTG AGT AGA GAC ACT GCT GCT 102 A I Q E A R R L L N L S R D T A AGAG ATG AAT GAA ACA GTA GAA GTC ATC TCA GAA ATG TTT GAC CTC CAG GAG 153 E M N E T V E V I S E M F D L Q ECCG ACC TGC CTA CAG ACC CGC CTG GAG CTG TAC AAG CAG GGC CTG CGG GGC 204 P T C L Q T R L E L Y K Q G L R GAGC CTC ACC AAG CTC AAG GGC CCC TTG ACC ATG ATG GCC AGC CAC TAC AAG 255 S L T K L K G P L T M M A S H Y KCAG CAC TGC CCT CCA ACC CCG GAA ACT TCC TGT GCA ACC CAG ATT ATC ACC 306 Q H C P P T P E T S C A T Q I I TTTT GAA AGT TTC AAA GAG AAC CTG AAG GAC TTT CTG CTT GTC ATC CCC 354 F E S F K E N L K D F L L V I PTTT GAC TGC TGG GAG CCA GTC CAG GAG GGT GGC GGT GGA TCT GGG GGT 402 F D C W E P V Q E G G G G S G GGGC GGC GGA TCC GCT CCC ATG ACC CAG ACA ACG CCC TTG AAG ACA AGC 450 G G G S A P M T Q T T P L K T STGG GTT AAC TGC TCT AAC ATG ATC GAT GAA ATT ATA ACA CAC TTA AAG CAG 501 W V N C S N M I D E I I T H L K QCCA CCT TTG CCT TTG CTG GAC TTC AAC AAC CTC AAT GGG GAA GAC CAA GAC 552 P P L P L L D F N N L N G E D Q DATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC CTG GAG GCA TTC AAC AGG 603 I L M E N N L R R P N L E A F N RGCT GTC AAG AGT TTA CAG AAC GCA TCA GCA ATT GAG AGC ATT CTT AAA AAT 654 A V K S L Q N A S A I E S I L K NCTC CTG CCA TGT CTG CCC CTG GCC ACG GCC GCA CCC ACG CGA CAT CCA ATC 705 L L P C L P L A T A A P T R H P ICAT ATC AAG GAC GGT GAC TGG AAT GAA TTC CGG AGG AAA CTG ACG TTC TAT 756 H I K D G D W N E F R R K L T F YCTG AAA ACC CTT GAG AAT GCG CAG GCT CAA CAG ACG ACT TTG AGC CTC GCG 807 L K T L E N A Q A Q Q T T L S L AATC TTT TGA 816 I F * ( 2 ) SEQ IDNO.2 ( ⅰ ) ：
(A) length: 271 amino acid
(B) type: amino acid
(C) chain: strand
(D) topological structure: linear (ⅱ) molecule type: polypeptide (ⅸ) sequence description: SEQ ID NO.2:APARSPSPST QPWEHVNAIQ EARRLLNLSR DTAAEMNETV EVISEMFDLQ 50EPTCLQTRLE LYKQGLRGSL TKLKGPLTMM ASHYKQHCPP TPETSCATQI 100ITFESFKENL KDFLLVIPFD CWEPVQEGGG GSGGGGGSAP MTQTTPLKTS 150WVNCSNMIDE IITHLKQPPL PLLDFNNLNG EDQDILMENN LRRPNLEAFN 200RAVKSLQNAS AIESILKNLL PCLPLATAAP TRHPIHIKDG DWNEFRRKLT 250FYLKTLENAQ AQQTTLSLAI F* 271
(2) information of SEQ ID NO:3:
(ⅰ) sequence signature:
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:3:
GCTCTA?GAG?GAGGATGTGG?CTGCAGA 27
(2) information of SEQ ID NO:4:
(ⅰ) sequence signature:
(A) length: 54 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:4:GCGGATCCGC CGCCACCCCC AGATCCACCG CCACCCTCCT GGACTGGCTC CCAG 54
(2) information of SEQ IDNO:5:
(ⅰ) sequence signature:
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:5:
CGGATCCGCT?CCCATGACCC?AGACAAC 27
(2) information of SEQ ID NO:6:
(ⅰ) sequence signature:
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:6:
CGGATCCGAA?CGAGCTGGAC?GTTGGAC 27
(2) information of SEQ ID NO:7:
(ⅰ) sequence signature:
(A) length: 31 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:7:
GCTCGAGAAA?AGAGCACCCG?CCCGCTCGCCC 31
(2) information of SEQ ID NO:8:
(ⅰ) sequence signature:
(A) length: 27 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ⅱ) molecule type: oligonucleotide
(ⅹ ⅰ) sequence description: SEQ ID NO:8:
CGGATCCGAA?CGAGCTGGAC?GTTGGAC 27
Claims (6)
1, the method for the fusion rotein MX of a kind of rHuGM-CSF of expression-secretion and interleukin is characterized in that, this method comprises:
(a) encoding sequence of rHuGM-CSF and the encoding sequence of interleukin are merged, form the polynucleotide sequence of encoding fusion protein MX;
(b) polynucleotide sequence with encoding fusion protein MX in the step (a) is inserted into expression vector, forms the expression vector that contains fusion rotein MX encoding sequence;
(c) change the expression vector that contains fusion rotein MX encoding sequence in the step (b) over to yeast cell;
(d) filter out the transformed yeast cells that the MX encoding sequence is integrated the people's gene group;
(e) under the condition that is fit to coding MX fusion rotein, transformed yeast cells in the culturing step (d);
(f) isolate the MX fusion rotein.
2, the method for claim 1 is characterized in that, this fusion rotein MX has the aminoacid sequence shown in the SEQ ID NO:2.
3, a kind of yeast cell, it is characterized in that, be integrated with the polynucleotide of the fusion rotein MX of the rHuGM-CSF of expression-secretion and interleukin in this zymic genome, and the fusion rotein of this nucleotide coding aminoacid sequence shown in SEQ ID NO:2.
4, yeast cell as claimed in claim 3 is characterized in that, this cell is selected from: yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and pichia pastoris phaff (Pichia pastoria).
5, yeast cell as claimed in claim 4 is characterized in that, this cell is pichia pastoris phaff (Pichia pastoria) MX CCTCC No.M99009.
6, the fusion rotein MX of a kind of rHuGM-CSF and interleukin is characterized in that, this albumen prepares with the described method of claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991168623A CN1148452C (en) | 1999-09-10 | 1999-09-10 | Yeast expression for secretory fusion protein |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB991168623A CN1148452C (en) | 1999-09-10 | 1999-09-10 | Yeast expression for secretory fusion protein |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1288059A true CN1288059A (en) | 2001-03-21 |
| CN1148452C CN1148452C (en) | 2004-05-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB991168623A Expired - Fee Related CN1148452C (en) | 1999-09-10 | 1999-09-10 | Yeast expression for secretory fusion protein |
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| CN (1) | CN1148452C (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308458C (en) * | 2002-11-06 | 2007-04-04 | 北京华特森基因科技有限公司 | Method for expressing human recombined interleukin 18 (rhIL-18) through yeast as well as product |
| WO2020024922A1 (en) * | 2018-07-30 | 2020-02-06 | 张晋宇 | Protein heterodimer and use thereof |
-
1999
- 1999-09-10 CN CNB991168623A patent/CN1148452C/en not_active Expired - Fee Related
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1308458C (en) * | 2002-11-06 | 2007-04-04 | 北京华特森基因科技有限公司 | Method for expressing human recombined interleukin 18 (rhIL-18) through yeast as well as product |
| WO2020024922A1 (en) * | 2018-07-30 | 2020-02-06 | 张晋宇 | Protein heterodimer and use thereof |
| US11795203B2 (en) | 2018-07-30 | 2023-10-24 | Jinyu Zhang | Protein heterodimer and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1148452C (en) | 2004-05-05 |
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