CN101089181A - Production method of recombination human interleukin-4 - Google Patents
Production method of recombination human interleukin-4 Download PDFInfo
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- CN101089181A CN101089181A CN 200610027656 CN200610027656A CN101089181A CN 101089181 A CN101089181 A CN 101089181A CN 200610027656 CN200610027656 CN 200610027656 CN 200610027656 A CN200610027656 A CN 200610027656A CN 101089181 A CN101089181 A CN 101089181A
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Abstract
This invention provides the coding sequence of recombination human interleukin-4, and method for producing said interleukin-4, and also the expression vactor and host cells used for said method. In this invention, pET system for expression of IL-4 is firstly used for obtaining high leven expression strain; and by controlling fermentation condition, the higher level of protein expression is obtained. Based on this and after large scale of research of purification of recombination human interleukin-4 of inclusion body expression, we obtain the method of purification of IL-4. By using this invention method, recombination human interleukin-4 protein can be obtained with high yield and high purity, to meet the clinical needs. This invention method is of high efficiency, simple and low cost.
Description
Technical field
The present invention relates to the genetically engineered field.More specifically, the invention provides the method for a kind of High-efficient Production recombinant human interleukin--4 (IL-4), comprise the structure of relevant engineering bacteria, recombinant human interleukin--4's expression and purifying process.
Background technology
Nineteen eighty-two, Howard finds in the T cells and supernatant a kind of somatomedin that promotes B cell proliferation to be arranged, originally called after Bcell growth factor-1 (B cell growth factor-1, BCSF-1).The laboratory that has claim B-cell stimulating factor (B cell stimulating factor-1, BSF-1), the T growth factor-2 (T cellgrowth-2, TCGF-2).Gene clone in 1986 success, and international uniform called after interleukin 4 (interleukin-4, IL-4).
IL-4 is the cytokine that is produced by helper cell (Th cell), mainly acts on the B cell, and it can activate the B cell that is in dormant state, promotes the propagation and the differentiation of B cell then, and IL-4 also plays an important role to IgG is synthetic.Can increase the humoral immunization of IgE mediation and the kill capability of killer cell.Discover that it can suppress the secretion of human monocyte IL-1B, TNF-α, IL-6 cytokine, reduce the expression of CD-5 in the B cell, promote the growth of human T cell.
Because IL-4 has the various biological function, its clinical indication is wider.IL-4 can be used for some tumor treatment; In some inflammation or autoimmune disease, therapeutic value is arranged, as be applied to arthritic treatment; IL-4 also may become the medicine of preventing and treating diabetes.The external at present reorganization IL-4 treatment tumour of using has entered I phase and II clinical trial phase.
Interleukin 4 is the approximately glycoprotein from 15kD to 19kD of molecular weight.The IL-4 precursor molecule is made up of 153 amino acid, and wherein the 1st～24 is signal peptide sequence.Sophisticated people IL-4 molecule is made up of 129 amino-acid residues, and theoretical relative molecular weight is 15000.Two possible glycosylation sites are arranged, can obtain 18-19KD through different N glycosylations, even 60KD.Have 6 Cys in the aminoacid sequence of mature peptide, lay respectively at the 3rd, 24,46,65,99,127 of mature peptide aminoacid sequence, (46-99), disulfide linkage is essential to the activity of IL-4 for 3-127,24-65 to contain 3 pairs of intramolecular disulfide bonds.
The configuration of IL-4 is based on α spiral (9～21,45～64,74～96,113～129).IL-4 is to multiple protein enzyme sensitivity, for example trypsinase, Chymotrypsin and V8 proteolytic enzyme.IL-4 can preserve in 4 ℃ more than 3 months, can preserve 1 month at room temperature, and 56 ℃ of 10min are not inactivated.In pH2～10, keep stable.
As if existing studies show that, glycosyl to the not influence of the biological activity of IL-4, the IL-4 of escherichia coli expression have with mammalian cell in the suitable biological activity of expressed products.Intestinal bacteria produce recombinant protein and have easy operation, growth rapidly and advantage such as the substratum requirement is simple.Expression and purifying work to IL-4 both at home and abroad has report, but expression amount is not high, generally accounts for the 5-10% of bacterial protein, and with the inclusion body formal representation, after inclusion body became renaturation, the target protein loss was very big, so that the IL-4 final yield is very low.
Therefore, press for the method for the new High-efficient Production recombinant human IL-4 of exploitation, the present invention has adopted escherichia expression system to express IL-4, by the optimization to people IL-4 gene, structure contains the people IL-4 expression carrier of optimization, and has obtained high-caliber expression strain; By the control fermentation condition, reached higher protein expression rate; On this basis the purifying of the rhIL-4 that inclusion body is expressed carried out a large amount of grope and study after, obtained high purity, high yield, can supply zooperal rhIL-4 albumen.
Summary of the invention
Purpose of the present invention just provides a kind of efficient and/or easy production recombinant human interleukin--4's method.
Another object of the present invention just provides the recombinant human interleukin--4's of optimization encoding sequence and the expression vector and the engineering strain that are used for this method.
In a first aspect of the present invention, just provided a kind of nucleotide sequence of coding recombinant interleukin-4 of optimization, it is characterized in that, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotide sequence is had any different shown in described nucleotide sequence coded district and the SEQ ID NO:1.
In a second aspect of the present invention, a kind of expression vector is provided, contain the described nucleotide sequence of claim 1.
In another preference, described expression vector is pET30a (+)/IL-4.
In a third aspect of the present invention, a kind of engineering cell is provided, it is characterized in that, contain the described expression vector of claim 2.
In another preference, described engineering cell is e. coli bl21 (DE3), is the genetic flaw bacterial strain of a plurality of proteolytic enzyme, and genotype is hsdSgal (a λ cIts857 ind1Sam7nim5lacUV5-T7 gene), is fit to very much efficiently expressing of foreign gene.
In a fourth aspect of the present invention, a kind of recombinant human interleukin--4's of production method is provided, the method comprising the steps of:
A) under the expression condition that is fit to, cultivate host cell as claimed in claim 4, thereby give expression to human interleukin-4 albumen; Preferably, described host cell is Bacillus coli cells BL21 (DE3).
B) separation and purification goes out the human interleukin-4 albumen of expression.
In another preference of the present invention, the culture condition of described step (a) comprising:
Cultivation is divided into cultivation stage and induction period, and it is about 5.0 that cultivation stage cultivation bacterial concentration reaches OD600, and induction time is 3～4hr, fermentation and inducing temperature remain on 37 ℃, the amount of trace element is 0.1-2ml/L, and the pH value of inductive phase is 7.0, and inductive phase, IPTG concentration was at 0.1-1mg/L.
In another preference of the present invention, the process of inducing can also be added casein hydrolysate (CA), peptone (peptone), milk powder, arginine etc. as protective material.
In another preference of the present invention, described step (b) comprises step:
I) fermented sample is removed supernatant by centrifugal and/or filter type, obtain fermentation back thalline;
Ii) the fermentation precipitation is carried out the broken bacterium of homogenate, washing acquisition inclusion body;
Iii) inclusion body is carried out the sex change dissolving;
Iv) chromatography purification, described chromatography is selected from: gel permeation chromatography, anion-exchange chromatography, cation-exchange chromatography and combination thereof.
V) adopt the method for dialysis renaturation, the renaturation supernatant carries out cation-exchange chromatography again and concentrates.
In another preference of the present invention, the inclusion body sex change after gel permeation chromatography, Q Sepharose F.F. chromatography, SP-Sepharose F.F. chromatography, dialysis renaturation, obtain after concentrating purity greater than 98%, yield is at the pure product more than 20%.
Description of drawings
The homology of theoretical sequence of Fig. 1 .IL-4 and IL-4 majorizing sequence relatively.Query: natural IL-4 gene order; Sbjct: the IL-4 gene order of optimization.
The structure route map of Fig. 2 .pET30a (+)/IL-4 expression plasmid.
Fig. 3. express the engineering bacterium expression form and determine.1. before inducing, after 2. inducing, 3,5. lysing cell supernatant, 4,6. lysing cell precipitation.
Fig. 4. the fermentation culture process thalli growth of engineering bacteria and target protein are expressed curve.
Fig. 5 .SP-Sepharose FF purifying electrophorogram.1.Marker, the supernatant of 2. dialysing, 3.SP-Sepharose FF ion exchange chromatography penetrates liquid, and 4-9.SP-Sepharose FF collects liquid.
Embodiment
The inventor is extensive studies by going deep into, by optimization design human interleukin-4 gene coded sequence, human interleukin-4 encoding sequence after optimizing is built into suitable expression vector, transformed host cell, obtain the high expression level bacterial strain, and by optimizing fermentation, purifying process, realized that human interleukin-4 efficiently expresses, and the human interleukin-4 that gives expression to keeps biologic activity.Finished the present invention on this basis.
The present invention is according to the human interleukin-4 natural acid sequence, press codon-bias, under the condition that does not change aminoacid sequence, optimize its nucleotide sequence, the synthetic proteic target gene sequences of recombinant human interleukin--4 of full gene through optimizing, with this gene clone in the pET3 (+) after the sequence verification, ordinary method with molecular cloning, construction of expression vector changes intestinal bacteria over to, goes out to express engineering cell by applying antibiotic-screening.The test tube screening obtains the high expression level engineering cell.Shake flask test shows, induce 3 hours after, target protein accounts for total protein more than 35%, more than the expression level 150mg/L.
After obtaining engineering cell, just can be under the condition that is fit to culturing engineering.In the present invention, engineering bacterium expression recombinant human interleukin--4's fermentation condition is not particularly limited.Can adopt the fermentation condition of this area routine.For example, suitable medium includes, but is not limited to following composition:
(vi) nitrogenous source contains organic nitrogenous source and inorganic nitrogen-sourced, wherein organic nitrogen source such as peptone, yeast extract powder, or the mixture of the two, total concn 0.5-1%; Inorganic nitrogen-sourced as ammoniacal liquor or (NH
4)
2SO
4Deng ammonium salt, concentration 0.35-25%.
(vii) inorganic salt comprise phosphoric acid salt, Ca
2+Salt, Mg
2+Salt etc.Preferably, phosphate buffer concentration 20-200mM, pH5-8; Mg
2+Salt 0-5mM.
(viii) in substratum, add VITMAIN B1 VITAMIN as a supplement, concentration 1～1000PPM.
(ix) according to M
9Trace element formula in the substratum, trace element can add 0.1-2ml/L training liquid.
(x) carbon source is a glucose, is single carbon source.Preferably, glucose concn is 1.0%, and glucose concn is 11.25% in the feed supplement.
For extensive acquisition recombinant human interleukin--4, need in fermentor tank, express cultivation.The present invention has studied pilot scale fermentation technology, and the expression level after the optimization reaches 500mg/L.
Behind fermentation expression, recombinant human interleukin--4's albumen of expressing is separated.Usually, the centrifugal acquisition fermented liquid postprecipitation of fermented sample elder generation is a thalline.The broken bacterium of homogenate obtains inclusion body through washing then, and the inclusion body sex change is after gel permeation chromatography, Q Sepharose F.F. chromatography, SP-sepharose F.F. chromatography, dialysis renaturation, concentrate etc.
Through different chromatography processes relatively, the purification process of optimization comprises:
1. fermented sample is carried out centrifugal acquisition fermentation postprecipitation;
By the inclusion body sex change after gel permeation chromatography, Q Sepharose F.F. chromatography, SP-sepharose F.F. chromatography, dialysis renaturation, concentrate, pure product purity is more than 98%, the about 120mg/L fermented liquid of yield.
The activity of pure product is measured by cytopathic-effect inhibition assay, and violet staining is measured OD with microplate reader
570Value is at last with mark Huaihe River, world product proofreading activity unit.Biological activity assay target protein specific activity reaches 2.5 * 10
6U/mg.
The recombinant human interleukin--4 can make various formulations with routine techniques behind the purifying.
In an example of the present invention, made up the escherichia coli expression engineering cell of producing the recombinant human interleukin--4, IPTG induces, and high-level inclusion body is expressed.
In another example of the present invention, the optimization of technology has by fermentation further improved recombinant human interleukin--4's expression amount.
In another example of the present invention, because the albumen inclusion body is expressed, thalline obtains inclusion body through homogenate, washing, can obtain pure product 120mg/L again behind a series of purification steps.
Stoste adds suitable auxiliary material behind the purifying, makes recombinant human interleukin--4's powder ampoule agent for injection.The preliminarily stabilised test shows that this powder injection is stable.
Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, and every liter of fermented liquid can obtain the pure product 120mg of recombinant human interleukin--4, is fit to industrialization production.
The invention has the advantages that:
What (1) select for use is e. coli bl21 (DE3) host cell, goes out the multiple copied transformant by applying antibiotic-screening, and then filters out the high expression level engineering cell.
(2) the recombinant human interleukin--4's protein gene after the optimization is highly suitable in e. coli bl21 (DE3) host cell and expresses, and has the characteristics of high expression level, high stable.
(3) by the crucial technological condition for fermentation of control, make expression level reach 500mg/L.
(4) because albumen is with the inclusion body formal representation, determined preferable operational path, the renaturation yield of metaprotein is improved greatly, the purifying process flow process is simple, can obtain highly purified IL-4 albumen, every liter of fermentor cultivation liquid can obtain more than 120 milligrams, purity is greater than 98% recombinant human interleukin--4.
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 normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
The structure of embodiment 1 expression plasmid and the acquisition of high expression engineering strain
According to the human interleukin-4 natural acid sequence, press codon-bias, under the condition that does not change aminoacid sequence, the complete proteic target gene sequences of gene synthesizing recombined human interleukin 4 (SEQ ID NO:1), the IL-4 gene order of this optimization and natural IL-4 gene order homology are 93% (see figure 1).Arrive among the pET-30a (+) this gene clone and sequence verification.
The expression vector establishment method is seen Fig. 2.Obtain purpose human interleukin-4 gene by pcr amplification, the PCR that handles gene IL-4 with Nde I and EcoRI double digestion respectively reclaims product and plasmid pET30a (+), and enzyme is cut the back corresponding fragment of recovery and spent the night 16 ℃ of connections with the T4 dna ligase.Connect product transformed into escherichia coli DH5 α, select resistance male clone on the LB flat board of kantlex containing, the extracting plasmid DNA, and carry out plasmid enzyme restriction and identify.Obtain in pET30a (+), having inserted recombinant plasmid pET30a (+)/IL-4 of IL-4 gene.
Expression plasmid pET30a (+)/IL-4 conversion that builds is entered e. coli bl21 (DE3) competent cell that has prepared, coating contains the LB flat board of 30 μ g/mL kantlex, transforming picking mono-clonal on the flat board then, with the alkaline lysis extracting and detect plasmid.The mono-clonal that conclusive evidence is contained expression plasmid pET30a (+)/IL-4 is preserved on the LB flat board that contains 30 μ g/mL kantlex.
Determining of embodiment 2 recombinant human interleukin--4s' expression and expression-form
Select expression engineering bacteria BL21 (DE3)/pET30a (+)/IL-4 that a strain is proved conclusively, cultivate and use the IPTG abduction delivering with the LB substratum, whether the SDS-PAGE electrophoresis detection has the appearance of purpose band, and analyzes its expression amount.The bacterium liquid of getting after a part is fermented is centrifugal, is resuspended in pH 8.0,20 mM Tris damping fluids, carrying out ultrasonic bacteria breaking under ice bath, and the ultrasonic cleer and peaceful ultrasound precipitation of centrifugal collection, the SDS-PAGE electrophoresis detection is determined its expression-form.Analyze its ultrasonic cleer and peaceful ultrasound precipitation, the target protein overwhelming majority shows that target protein is with inclusion body formal representation (see figure 3) in precipitation.
Get expression engineering bacteria BL21 (DE3)/pET30a (+)/rhIL-4 that is accredited as positive colony, containing 30ug/ml kantlex LB cultivation 14～16 hours; Be inoculated in by 3% inoculum size again and grow to OD among the LB
600Be 1～3, as secondary seed solution.(Glucose 1%, Yeast extract 0.5%, K for preparation improvement M9 fermention medium 2.96L
2HPO
10.5%, KH
2PO
40.35%, (NH
4)
2HPO
40.35%, MgSO
40.025%, CaCL
20.1%), pours in the fermentor tank that cleaned, even behind jar autoclaving, be cooled to 30～40 ℃, cultured secondary seed solution is inserted fermentor tank, 37 ℃ of cultivations by 1.5% inoculum size, and constantly adjust ventilation flow rate and mixing speed according to oxygen dissolving value, the control dissolved oxygen is more than 40%.Per hour OD is surveyed in sampling
600Value.Be cultured to OD
600Reach about 5.0 and begin to induce, add IPTG and induce to final concentration 1mM in fermentor tank, induce and received in 3.5 hours jar, fermented liquid is in 4200rpm, and 20 minutes centrifugal, and collecting precipitation is weighed.Expression product accounts for 35% of bacterial protein.See Fig. 4.
Thalline after the fermentation is resuspended in 20 mM Tris, pH 8.0, damping fluid, the broken bacterium of high-pressure homogenization under ice bath, centrifugal collecting precipitation is used the washing precipitation of Tris damping fluid again, obtain inclusion body, in 1: 10 ratio, with inclusion body sex change dissolving in the 6M guanidine hydrochloride solution, the protein solution centrifuging and taking supernatant after the sex change; To containing the 5% sucrose damping fluid dialysis of 1mM reduced glutathion and 0.1mM Sleep-promoting factor B, make the target protein renaturation; Supernatant after the renaturation is crossed SP-Seharose fast flow ion exchange column, with 0-1M NaCl buffer solution for gradient elution, is in charge of and collects the eluted protein peak; After the dialysis of SP elution peak, cross Q-Sepharose fast flow ion exchange column, concentrate.The purity and the content (see figure 5) of 15%SDS-PAGE electrophoresis detection purge process target protein.
Through above purifying process, can get the pure product 120mg/L of albumen at last.
Personnel selection erythrocyte leucocythemia cell (TF-1) is pressed reference 9 methods and is detected people IL-4, this method detects activity (Wang Junzhi (Wang Junzhi) the .Research Exploitation and QualityControl of Biotechnic Medicament that IL-4 promotes T cell proliferation, 2002, Science Press).The activity of the recombination human interleukin of purifying-4 reaches 2.5 * 10
6U/mg.
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
＜110〉the new source of students in Shanghai Biopharmaceutical Research Inc.
＜120〉recombinant human interleukin--4's production method
<160>2
<210>1
<211>498
<212>DNA
＜213〉artificial sequence
<221>misc_feature
<222>(1).(498)
＜223〉recombinant human interleukin--4's gene of You Huaing
<400>1
atgagttaca?acttgcttgg?attcctacaa?agaagcagca?attttcagtg?tcagaagctc?60
ctgtggcaat?tgaatgggag?gcttgaatac?tgcctcaagg?acaggatgaa?ctttgacatc?120
cctgaggaga?ttaagcagct?gcagcagttc?cagaaggagg?acgccgcatt?gaccatctat?180
gagatgctcc?agaacatctt?tgctattttc?agacaagatt?catctagcac?tggctggaat?240
gagactattg?ttgagaacct?cctggctaat?gtctatcatc?agattaacca?tctgaagaca?300
gtcctggaag?aaaaactgga?gaaagaagat?ttcaccaggg?gaaaactcat?gagcagtctg?360
cacctgaaaa?gatattatgg?gaggattctg?cattacctga?aggccaagga?gtacagtcac?420
tgtgcctgga?ccattgtcag?agtggaaatc?ctaaggaact?tttacttcat?taacagactt?480
acaggttacc?tcagaaac 498
<210>2
<211>166
<212>PRT
＜213〉homo sapiens
<400>2
Met?Ser?Tyr?Asn?Leu?Leu?Gly?Phe?Leu?Gln?Arg?Ser?Ser?Asn?Phe
1 5 10 15
Gln?Cys?Gln?Lys?Leu?Leu?Trp?Gln?Leu?Asn?Gly?Arg?Leu?Glu?Tyr
20 25 30
Cys?Leu?Lys?Asp?Arg?Met?Asn?Phe?Asp?Ile?Pro?Glu?Glu?Ile?Lys
35 40 45
Gln?Leu?Gln?Gln?Phe?Gln?Lys?Glu?Asp?Ala?Ala?Leu?Thr?Ile?Tyr
50 55 60
Glu?Met?Leu?Gln?Asn?Ile?Phe?Ala?Ile?Phe?Arg?Gln?Asp?Ser?Ser
65 70 75
Ser?Thr?Gly?Trp?Asn?Glu?Thr?Ile?Val?Glu?Asn?Leu?Leu?Ala?Asn
80 85 90
Val?Tyr?His?Gln?Ile?Asn?His?Leu?Lys?Thr?Val?Leu?Glu?Glu?Lys
95 100 105
Leu?Glu?Lys?Glu?Asp?Phe?Thr?Arg?Gly?Lys?Leu?Met?Ser?Ser?Leu
110 115 120
His?Leu?Lys?Arg?Tyr?Tyr?Gly?Arg?Ile?Leu?His?Tyr?Leu?Lys?Ala
125 130 135
Lys?Glu?Tyr?Ser?His?Cys?Ala?Trp?Thr?Ile?Val?Arg?Val?Glu?Ile
140 145 150
Leu?Arg?Asn?Phe?Tyr?Phe?Ile?Asn?Arg?Leu?Thr?Gly?Tyr?Leu?Arg
155 160 165
Asn
166
Claims (10)
1. the recombinant human interleukin--4 that encodes dna sequence dna is characterized in that, the sequence shown in its dna sequence dna and the SEQID NO:1 is had any different, but the aminoacid sequence of coding shown in SEQ ID NO:2.
2. an expression vector is characterized in that, it contains the described dna sequence dna of claim 1.Preferably, described expression vector is pET30a (+)/IL-4.
3. a host cell is characterized in that, it contains described expression vector of claim 2 or the described coding of claim 1 recombinant human interleukin--4's dna sequence dna.
4. method of producing the recombinant human interleukin--4 is characterized in that it comprises step:
A) under the expression condition that is fit to, cultivate host cell as claimed in claim 4, thereby give expression to human interleukin-4 albumen; Preferably, described host cell is Bacillus coli cells BL21 (DE3).
B) separation and purification goes out the human interleukin-4 albumen of expression.
5. method as claimed in claim 4 is characterized in that described host cell is intestinal bacteria.
6. method as claimed in claim 4 is characterized in that, carries out the high-density suspension culture in step (a).
7. method as claimed in claim 4 is characterized in that, described step (b) comprises step:
(i) fermented sample is removed supernatant by centrifugal and/or filter type, obtain fermentation back thalline;
(ii) the fermentation precipitation is carried out the broken bacterium of homogenate, washing acquisition inclusion body;
(iii) inclusion body is carried out the sex change dissolving
(iv) chromatography purification, described chromatography is selected from: gel permeation chromatography, anion-exchange chromatography, cation-exchange chromatography and combination thereof.
(v) adopt the method for dialysis renaturation, the renaturation supernatant carries out cation-exchange chromatography again and concentrates.
8. method as claimed in claim 4 is characterized in that, described culture condition is: culture temperature is 30-40 ℃, more preferably 34-38 ℃; PH is between 6～8, more preferably between 6.5～7.5; Glucose or glycerol concentration are 0.1～5%, more preferably 1.0%; Add VITMAIN B1 VITAMIN as a supplement; Trace element can add 0.1-2ml/L training liquid; Inductor be IPTG concentration between 0.1-1mg/L, induce before OD600 between 0.5～20, more preferably between 1～10; Induction time 0.5～10 hour, more preferably 1～5 hour.
9. method as claimed in claim 4 is characterized in that, described purification condition is: fermented sample is carried out centrifugal acquisition fermentation postprecipitation; The precipitation sex change obtains the pure product of purity more than 98% after gel permeation chromatography, Q Sepharose F.F. chromatography, SP-Sepharose F.F. chromatography, dialysis renaturation, concentrate.
10. the recombinant human interleukin--4 comprises the host cell that transforms with the described expression vector of claim 2, cultivates transformant, obtains the method preparation of recombinant human interleukin--4's step from culture.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320447A (en) * | 2013-05-20 | 2013-09-25 | 深圳市亚太兴实业有限公司 | Recombinant human interleukin 4 gene, and fermentation method of engineering bacteria comprising recombinant human interleukin 4 gene |
| CN108218978A (en) * | 2016-12-13 | 2018-06-29 | 何伟 | A kind of recombinant interleukin 18 and preparation method and application |
| CN110818789A (en) * | 2018-08-07 | 2020-02-21 | 三生国健药业（上海）股份有限公司 | Purification method of high-purity cynomolgus monkey interleukin 17A |
-
2006
- 2006-06-13 CN CN 200610027656 patent/CN101089181A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103320447A (en) * | 2013-05-20 | 2013-09-25 | 深圳市亚太兴实业有限公司 | Recombinant human interleukin 4 gene, and fermentation method of engineering bacteria comprising recombinant human interleukin 4 gene |
| CN108218978A (en) * | 2016-12-13 | 2018-06-29 | 何伟 | A kind of recombinant interleukin 18 and preparation method and application |
| CN108218978B (en) * | 2016-12-13 | 2020-05-12 | 沈阳何氏眼产业集团有限公司 | Recombinant interleukin 18 and preparation method and application thereof |
| CN110818789A (en) * | 2018-08-07 | 2020-02-21 | 三生国健药业（上海）股份有限公司 | Purification method of high-purity cynomolgus monkey interleukin 17A |
| CN110818789B (en) * | 2018-08-07 | 2023-02-28 | 三生国健药业（上海）股份有限公司 | Purification method of high-purity cynomolgus monkey interleukin 17A |
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