CN1844392A - Recombinant human ciliary nerve trophic factor mutant and method for preparing same - Google Patents
Recombinant human ciliary nerve trophic factor mutant and method for preparing same Download PDFInfo
- Publication number
- CN1844392A CN1844392A CN 200610076719 CN200610076719A CN1844392A CN 1844392 A CN1844392 A CN 1844392A CN 200610076719 CN200610076719 CN 200610076719 CN 200610076719 A CN200610076719 A CN 200610076719A CN 1844392 A CN1844392 A CN 1844392A
- Authority
- CN
- China
- Prior art keywords
- rhcntf
- damping fluid
- mutant
- lactose
- chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relate to recombination protein and Process for preparing, concretely, relate to recombine human ciliary's neurotrophy factor (abbreviation rhCNTF) mutant and Process for preparing, more concretely, the invention relate to lactose evoked rhCNTF mutant highly effective soluble expression in bacillus coli BL21 (DE3), the method of separation and purification and prepare rhCNTF mutant though the method.
Description
Technical field
The present invention relates to recombinant protein and its preparation method, be specifically related to recombination human ciliary neurotrophy factor (being called for short rhCNTF) mutant and preparation method thereof, in particular, the present invention relates to lactose-induced rhCNTF mutant efficient soluble-expression, and method of separation and purification in addition therefrom in e. coli bl21 (DE3), and the rhCNTF mutant by this method preparation.
Background technology
Ciliary neurotrophic factor (CNTF) is to extract from the ciliary body of chicken at first, can keep the survival of chicken parasympathetic ganglion, and therefore gains the name.The CNTF gene was cloned and was expressed early than 1989.Natural CNTF is a protein of being made up of 200 amino-acid residues, no intramolecular disulfide bond, and no secretion signal, the sugar based site, its higher structure prediction has similar auger frame shelf structure with IL-6, LIF etc.CNTF has multiple function, can impel multiple neuron survival, be that first discovery can be kept in the body and the survival of stripped dynamoneure and the neurotrophic factor of enation, CNTF also has the muscle nutrition effect, in nervous system development, differentiation and nerve injury reparation, have important effect, can be used for preparing the medicine of treatment Mammals central nervous system injury, peripheral nerve injury, peripheral nerve disease etc.
For the expression of recombinant protein in intestinal bacteria, the lac promotor is the promotor of using always, and IPTG has high inducibility to this promotor, but because of its expensive and potential toxicity, makes its application in scale operation be subjected to great restriction.Lactose replaces IPTG not only can eliminate above-mentioned restriction as inductor, and can reach near IPTG inductive expression level.At present, people are usually after interpolation lactose in nutrient solution is as inductor, with the main carbon source of lactose as the target protein expression phase.Because lactose is in intravital mechanism of action of bacterium and characteristics, induce the back under than the high-cell density situation, with the main carbon source of lactose, finally do not cause the thalline final concentration high and thalline harvest yield is on the low side as genetic engineering bacterium.
At present, how escherichia coli expression CNTF is with the inclusion body form in this area, and when foreign gene efficiently expressed in intestinal bacteria, expression product often flocked together in cell, forms the refractive power corpusculum of size 0.5～1 μ m, i.e. inclusion body.For example, (horse blood rain, Zhu Meicai, Cai Qing such as horse blood rain, account for will, the CNTF engineering bacteria is cultivated and product purification. and the Chinese biological goods are learned magazine, calendar year 2001, Vol.14 No.3) utilizes engineering bacteria DH5 α/pBV220CNTF to carry out the expression study of CNTF, and it is the expression of inclusion body form.Pass through computer aided design (CAD) among the Chinese patent application No.02136033.2 of Jiuyuan Gene Engineering Co., Ltd., Hangzhou, gene to rhCNTF is modified transformation, obtain the cDNA of mutant rhCNTF gene, this cDNA of synthetic, construction recombination plasmid, transformed into escherichia coli is expressed the inclusion body that obtains rhCNTF through the automatic fermenter high-density and is expressed.Disclosed rhCNTF also is with the inclusion body formal representation in intestinal bacteria among the US 6602687 in addition.
Though it is higher that inclusion body is expressed general expression amount, but after inclusion body formed, the primary structure of these inclusion body internal proteins was correct, but not through correctly being folded to form natural higher structure, thereby the shortage biologic activity, need increase sex change, renaturation step to recover its native conformation.Proteinic annealing issues is still a difficult point in the biotechnology at present, and the renaturation workload is big, and renaturation yield is low, the cost height.As described in embodiment among the US 6602687, expression level and wet thallus total concn according to estimation are calculated, every gram wet thallus can obtain 15～45mg CNTF in theory, but through behind inclusion body sex change renaturation and the purifying, final pure product harvest yield only is every gram wet thallus 6mg CNTF.
Li Hong equalization (Li Hongjun, Ma Yanbing, Su Ting etc. the Human Ciliary Neurotrophic Factor clone, express, purifying and biological activity thereof. the Chinese biological goods are learned magazine, 2002, Vol.15, No.3) research recombinant expression plasmid PBV220-CNTF has obtained nonfused stability and high efficiency expression in e. coli bl21, by the thin layer scanning analysis, target protein accounts for 45% of bacterial protein, the rhCNTF that expression is all arranged in ultrasonic supernatant and precipitation, about 60% exists with soluble form, and solvable supernatant has been carried out a simple step cation-exchange chromatography purifying, and this is uniquely in the state of the art to mention the report that recombinant human CNTF expresses with soluble form.At present still be not raw material, be the purification process of purpose with a large amount of preparation high purities and high activity recombinant human ciliary neurotrophic factor with solvable supernatant.
Therefore, it is the method that purpose prepares recombination human ciliary neurotrophy factor with low-cost, high yield that this area presses for exploitation, and described preparation comprises expression, separation and the purifying of recombination human ciliary neurotrophy factor.
Summary of the invention
Therefore, an object of the present invention is to provide the method for solubility expression high reactivity rhCNTF.Wherein said solubility and high reactivity are realized by following sudden change: the halfcystine that rhCNTF is the 17th replaces with L-Ala or arginine; The 63rd L-glutamic acid replaces with arginine; The 64th Histidine replaces with L-Ala; Remove 1～30 amino acid of C-terminal of rhCNTF; Or the combination of these sudden changes.C-terminal amino acid is preferably removed 13 or 15.Described sudden change makes up that preferred the 17th halfcystine replaces with L-Ala, the 63rd L-glutamic acid replaces with arginine and C-terminal is removed 15 amino acid whose combinations.
Another object of the present invention provides the derivational expression method of high reactivity rhCNTF.Wherein said abduction delivering adds glucose after lactose-induced again and realizes as main carbon source by adding.Described adding lactose is included in and adds lactose in the initial medium, adds lactose and add lactose in the additional liquid of carbon source in the additional liquid of nitrogenous source.The final concentration that wherein adds lactose is 1 weight %～2 weight %, be preferably 1.7 weight %, induced about 1～4 hour, slowly add glucose after preferred 2～2.5 hours again as main carbon source, and control culture temperature be 25～28 ℃, control nutrient solution in dissolved oxygen value be 10～15%.
Another object of the present invention provides the separation purification method of high reactivity rhCNTF, and it comprises saturation ratio 20～45% grade ammonium sulfate salting-outs, hydrophobic chromatography, anion-exchange chromatography and gel-filtration purifying, and the purity of acquisition is greater than 98%.
In one embodiment of the invention, the method for preparing solubility rhCNTF comprises the following steps:
(a) rhCNTF mutant gene sequence clone is gone into expression vector obtaining recombinant vectors, and import in the intestinal bacteria, generate the genetic engineering bacterium that contains recombinant vectors;
(b) the culturing gene engineering bacteria is to A
600=12～14 o'clock, add lactose, induce rhCNTF mutant solubility expression in genetic engineering bacterium;
(c) induce after, in nutrient solution, slowly add glucose as main carbon source, and dissolved oxygen value is 10～15% in the control nutrient solution, continues to cultivate;
(d) treat the A of genetic engineering bacterium
600=20～30 o'clock, gather in the crops thalline, and bacterial cell disruption is obtained brokenly bacterium liquid;
(e) the broken bacterium liquid of purifying obtains purified product.
In a preferred embodiment of the invention, above-mentioned intestinal bacteria are e. coli bl21 (DE3).
In another preferred embodiment of the present invention, the sudden change that relates in the described rhCNTF mutant is selected from: the halfcystine that rhCNTF is the 17th replaces with L-Ala or arginine; The 63rd L-glutamic acid replaces with arginine; The 64th Histidine replaces with L-Ala; Remove 1～30 amino acid of C-terminal of rhCNTF; Or the combination of these sudden changes.
In a preferred embodiment of the present invention, C-terminal amino acid is preferably removed 13 or 15.
In another more preferred of the present invention, described sudden change makes up that preferred the 17th halfcystine replaces with L-Ala, the 63rd L-glutamic acid replaces with arginine and C-terminal is removed 15 amino acid whose combinations.
In a preferred embodiment of the invention, described bacterial cell disruption uses high pressure homogenizer to carry out, and condition is 800bar, 2 circulations.
In a preferred embodiment of the invention, the A of genetic engineering bacterium in the step (d)
600=25.
In a preferred embodiment of the invention, in the step (e) broken bacterium liquid being carried out saturation ratio successively is 20%～45% grade ammonium sulfate salting-out, hydrophobic chromatography, anion-exchange chromatography and gel-filtration purifying, obtains purified product.
In a preferred embodiment of the invention, described hydrophobic chromatography filler is Butyl Sepharose 4 FastFlow or Phenyl Sepharose 4 Fast Flow, in the chromatography process, use the damping fluid balance chromatography column that contains ammonium sulfate, behind the last sample, with the assorted peak of the buffer solution elution of sulfur acid ammonium, use the not active peak of buffer solution elution of sulfur acid ammonium more earlier.In a preferred embodiment of the present invention, the ammonium sulfate concentrations scope that above-mentioned level pad contains is 0.5～1.0mol/L; Washing the ammonium sulfate concentrations scope that assorted peak damping fluid contains is 0.2～0.3mol/L; Damping fluid is Tris-HCl, and concentration range is 10～50mmol/L, and the pH value is 7.0～9.0.
In a preferred embodiment of the invention, described anion-exchange chromatography filler is Q SepharoseFast Flow or DEAE Sepharose Fast Flow, use the damping fluid balance chromatography column that contains salt in the chromatography process, behind the last sample, earlier with the assorted peak of saliniferous buffer solution elution, again with containing the more active peak of buffer solution elution of high salt concentration.In a preferred embodiment of the present invention, the damping fluid of use is the Tris-HCl damping fluid, and concentration is 20～50mmol/L, and the pH value is 7.5～8.5.In a more preferred of the present invention, the salt that contains in the damping fluid is sodium-chlor, the sodium chloride concentration that contains in the damping fluid at the assorted peak of the first step wash-out is 20～50mmol/L, and the sodium chloride concentration that contains in the damping fluid at the active peak of the second step wash-out is 0.13～0.20mol/L.
In another embodiment of the invention, the preparation method of rhCNTF mutant of the present invention also comprises the step that concentrates activated protein solution by ammonium sulfate precipitation between anion-exchange chromatography and gel permeation chromatography step.
In an embodiment preferred of the present invention, the filler of described gel permeation chromatography is Superdex 75prep grade or Sephacryl S-200 High Resolution, use phosphate buffered saline buffer to carry out balance and wash-out, concentration is 5～50mmol/L, and the pH value is 7.0～8.5.
In addition, the invention still further relates to the rhCNTF mutant of the biologically active for preparing according to above-mentioned method.
Description of drawings
Figure 1 shows that rhCNTF mutant SDS-PAGE with the IPTG abduction delivering in e. coli bl21 (DE3) schemes under the shake-flask culture condition; No. 1 to No. 7 sample is followed successively by does not induce brokenly bacterium liquid supernatant, induce broken bacterium liquid supernatant after 1 hour, induce broken bacterium liquid supernatant after 2 hours, induce broken bacterium liquid supernatant after 3 hours, induce broken bacterium liquid supernatant after 4 hours, induce broken bacterium liquid precipitate after 4 hours, induce the full bacterium liquid after 4 hours, every swimming lane is the sample that obtains after the equivalent fermenation raw liquid is handled.M is the molecular weight of albumen standard, and band is followed successively by 116kD, 66kD, 45kD, 34kD, 25kD, 18kD, 14kD from big to small.If no special instructions, the SDS-PAGE electrophoresis of the following drawings all uses same molecular weight of albumen standard.
Figure 2 shows that rhCNTF mutant SDS-PAGE with lactose-induced expression in e. coli bl21 (DE3) schemes under the shake-flask culture condition; No. 1 to No. 8 sample is followed successively by does not induce brokenly bacterium liquid supernatant, induce broken bacterium liquid supernatant after 1 hour, induce broken bacterium liquid supernatant after 2 hours, induce broken bacterium liquid supernatant after 3 hours, induce broken bacterium liquid supernatant after 4 hours, induce broken bacterium liquid supernatant after 5 hours, induce broken bacterium liquid supernatant after 6 hours, induce the full bacterium liquid after 6 hours, every swimming lane is the sample that obtains after the equivalent fermenation raw liquid is handled.M is the molecular weight of albumen standard.
Figure 3 shows that rhCNTF mutant SDS-PAGE with lactose-induced expression in e. coli bl21 (DE3) schemes under the fermentor cultivation condition.No. 1 to No. 9 sample is followed successively by the broken bacterium liquid supernatant of inducing back sampling in 1～9 hour.
Figure 4 shows that SDS-PAGE (13.5%) analytical results of purge process gained main ingredient, coomassie brilliant blue staining, sample are 1 in proper order: broken bacterium liquid supernatant; 2: through the broken bacterium liquid supernatant of 20% saturation ratio ammonium sulfate precipitation, 3: through the broken bacterium liquid supernatant of 20%～45% saturation ratio ammonium sulfate precipitation; 4: through the broken bacterium liquid supernatant of 20%～45% saturation ratio ammonium sulfate precipitation; 5:Butyl hydrophobic chromatography target peak; 6:Q anion-exchange chromatography target peak; 7:Superdex75 gel permeation chromatography target peak; M: molecular weight of albumen standard.1～7 track applied sample amount is respectively 30 μ g, 30 μ g, 8 μ g, 40 μ g, 20 μ g, 17 μ g, 15 μ g.
Figure 5 shows that final pure product SDS-PAGE picture and purity check result.1, No. 2 swimming lane applied sample amount among the SDS-PAGE is respectively 10 μ g, and 15 μ g, M are the molecular weight of albumen standard.Thereafter chart is according to the electrophoresis picture, the Labwarks that is provided by UVP company
TMThe result that target protein content obtains in the Analysis Software software analysis track.
Fig. 6 is according to the resulting dose-effect curve of the biological activity determination method of embodiment 6.
Embodiment
In the context of the present specification, unless specialize, otherwise the used any technical term of this specification sheets has those of ordinary skills' implication of common sense in the art, and the experimental technique of unreceipted actual conditions is according to the normal experiment method, translate molecular cloning experiment guide second edition, Science Press as Jin Dongyan etc., Beijing, 1992; Wang Jiazheng etc., protein technical manual, Science Press, Beijing, 2001; The thick plinth of Zhu etc. is translated, protein purification and identification experiment guide, scientific publication Du, Beijing, 1999; With the Li Yuyang chief editor, gene expression technique, Science Press, Beijing, 2000 described methods are carried out; Or the process specifications of being advised according to peace agate West Asia company carries out.
In one embodiment of the invention, at first naturally occurring hCNTF aminoacid sequence is suddenlyd change more stable to obtain, active higher, the rhCNTF mutant that can express in the solubility mode.Described sudden change is selected from: the halfcystine that rhCNTF is the 17th replaces with L-Ala or arginine; The 63rd L-glutamic acid replaces with arginine; The 64th Histidine replaces with L-Ala; Remove 1～30 amino acid of C-terminal of rhCNTF; Or the combination of these sudden changes.
Wherein, the 17th halfcystine by listed aminoacid replacement after, can avoid intermolecular disulfide linkage, belong to experimental result for better stability is arranged, under physiological pH, temperature, be more conducive to the performance of active function, to the preparation medicament be significant.
The L-glutamic acid that rhCNTF is the 63rd by listed aminoacid replacement after, mutant protein can be more consumingly in stimulated in vitro neuron survival and differentiation, show as external that active to detect index better, and show as the raising of the experimental result of animal model in vivo.
The Histidine that rhCNTF is the 64th by listed aminoacid replacement after, mutant protein biological activity has in vivo had and has significantly improved.
After one terminal amino acid of the C-terminal of rhCNTF is removed, preferably remove 13 and 15 amino acid after, mutant protein has better solubleness and stability under physiological status.
In above-mentioned sudden change, will be referred to preferably that the 17th halfcystine replaces with L-Ala, the 63rd L-glutamic acid replaces with arginine and C-terminal is removed 15 amino acid whose sudden changes combinations, the rhCNTF mutant that possesses above-mentioned three kinds of sudden changes simultaneously, be called rhCNTF-TS, this advantage that can gather its each mutant to a certain extent that is combined in can increase significantly than the rhCNTF molecule of natural acid sequence in stability, biological activity.
Used in the present invention term " mutant or rhCNTF mutant " unless otherwise indicated, promptly refer to contain the rhCNTF molecule of said mutation site or its combination, its all be with natural hCNTF aminoacid sequence (GenBank GI:25952136) serve as the sudden change object.The method that obtains the said mutation body is known in those skilled in the art.
Exogenous gene sequence is inserted suitable expression vector and imports colibacillary method is as well known to those skilled in the art, for example translates molecular cloning experiment guide second edition, Science Press, Beijing, 1992 referring to Jin Dongyan etc.After obtaining containing the bacterial strain of recombinant vectors, under 37 ℃, bacterial strain is cultivated with substratum such as LB, after bacterial growth arrives certain density (as, A
600=0.6 o'clock), can in substratum, add finite concentration (as 1mmol/L) IPTG or (as 2%) lactose, bacterium is under the inducing of inductor, can great expression rhCNTF or mutant protein, we obtain these bacteria samples, just can to rhCNTF and or mutant protein purify and study.
In explanation of the present invention and embodiment, estimation about purity of protein and content, if no special instructions, all be to be analytic target with the SDS-PAGE picture, use electrophoresis picture analysis software to calculate to get, such software is that (the LabworksTM analysis software that provides as UVP company) can be provided or buy the public.
In one embodiment of the invention, use lactose, the solubility expression that preferably uses 1～2% lactose to induce rhCNTF as inductor.Since lactose-induced key be when inducing in the nutrient solution concentration of glucose enough low, therefore between inductive phase, lactose is both as the inductor of expression of recombinant proteins, again as main carbon source.But, consider that lactose can limit the final concentration and the thalline harvest yield of thalline as carbon source, make thalline final concentration and thalline harvest yield on the low side, in addition, as carbon source, the price of lactose is comparatively expensive, and bioavailability is lower again, thereby causes production cost to improve.Therefore, the present invention preferably slowly add glucose as main carbon source after inducing about 2～2.5 hours, and the control culture temperature is 25～28 ℃ after inducing for some time, dissolved oxygen value is 10～15% in the control nutrient solution, and target protein as much as possible is expressed with soluble form.
As a result, add glucose after lactose-induced for some time and not only can significantly improve thalline harvest yield, and do not influence the soluble-expression of target protein at all with other nitrogenous sources.Target protein forms soluble-expression more than 90%, soluble-expression amount (ratio that the target protein of solubility accounts in all solubility tropinas) reaches about 20%.
In the amplification culture experiment, the present invention adopts semisynthetic medium in fermentor tank, adds carbon source (as glucose) and the additional liquid (as Tryptones, yeast extract and casein hydrolysate) of nitrogenous source by stream before inducing, and reaches A at cell concentration
600=12～14 o'clock, add final concentration and be 1～2% lactose and induce, stop stream and add carbon source and replenish liquid, continue stream and add nitrogenous source and replenish liquid.Induced the back 2～2.5 hours at interval, and recovered stream and add the additional liquid of carbon source.Control pH value 6.6～7.0 in the whole culturing process, dissolved oxygen value 30%～50% before inducing, 37 ℃ of temperature are induced back dissolved oxygen value 5%～20%, begin stream after inducing and add behind the additional liquid of carbon source 20～30 ℃ of controlled temperature.More preferably, control pH value 6.8 in the whole culturing process, dissolved oxygen value 40% before inducing is induced back dissolved oxygen value 10%～15%, begins stream after inducing and adds behind the additional liquid of carbon source 25～28 ℃ of controlled temperature.Add the flow acceleration that replenishes liquid by in good time adjustment stream, control bacterium specific growth rate (definition of specific growth rate referring to: Zhang Yuanxing writes, bio-reactor engineering, P37, press of East China University of Science, calendar year 2001) is 0.2～0.3h before inducing
-1, be 0.05～0.15h after inducing
-1Induce back 6 hours results thalline, bacterial density can reach A
600About=25, wet thallus output 50g/L, the target protein more than 90% forms solubility expression, and the soluble-expression amount is about 20% (Fig. 2).
The bacterial cell disruption of fermentation results is obtained brokenly bacterium liquid, then broken bacterium liquid is carried out grade ammonium sulfate salting-out, hydrophobic chromatography, anion-exchange chromatography and gel-filtration purifying successively, obtain the high also recombination human ciliary neurotrophy factor of biologically active of purity.
With the wet thallus of the fermentation results ratio by 1: 10, with PBS or the resuspended thalline of Tris-HCl damping fluid, through the broken bacterium of high pressure homogenizer, the broken bacterium condition optimization of high pressure homogenizer breaks 2 circulations of bacterium under the pressure of 800bar.
The grade ammonium sulfate salting-out that broken bacterium liquid is carried out is to remove bacterial chip and most of foreign protein, and grade ammonium sulfate salting-out is preferably collected the fractionation precipitation part of 20%～45% saturation ratio.
The hydrophobic chromatography filler is Butyl Sepharose 4 Fast Flow, can also be Phenyl Sepharose 4 FastFlow, 10～50mmol/L Tris-HCl damping fluid balance the chromatography column that contains 0.5～1mol/L ammonium sulfate that uses, the pH value of damping fluid is 7.0～9.0, then with level pad dissolving ammonium sulfate precipitation sample, on the sample behind the hydrophobic chromatography post, earlier with the 10～50mmol/L Tris-HCl that contains 0.2～0.3mol/L ammonium sulfate, the buffer solution elution of pH7.0～9.0 peak of mixing, use not 10～50mmol/L Tris-HCl of sulfur acid ammonium again, the active peak of the buffer solution elution of pH7.0～9.0.
The anion-exchange chromatography filler is Q Sepharose Fast Flow, can also be DEAE Sepharose FastFlow, the level pad that uses in anion-exchange chromatography is the Tris-HCl damping fluid, concentration is 20～50mmol/L, the pH value of damping fluid is 7.5～8.5, active peak specific conductivity through hydrophobic chromatography is lower, directly go up the anion-exchange chromatography post, behind the last sample, earlier with the assorted peak of saliniferous buffer solution elution, again with containing the more active peak of buffer solution elution of high salt concentration, damping fluid is selected the Tris-HCl damping fluid for use, concentration is 20～50mmol/L, and the pH value of damping fluid is 7.5～8.5, and the salt that damping fluid contains can be sodium-chlor, also can be sodium sulfate, ammonium sulfate, preferred sodium-chlor, the sodium chloride concentration that contains in the damping fluid at the assorted peak of the first step wash-out is 0.02～0.05mol/L, the sodium chloride concentration that contains in the damping fluid at the active peak of the second step wash-out is 0.13～0.2mol/L.
Behind anion-exchange chromatography, use the ammonium sulfate precipitation method to concentrate active peak, last is the phosphate buffered saline buffer counter-balanced gel permeation chromatography post of 5～50mmol/L, pH value 7.5～8.5 with concentration, the gel permeation chromatography filler is Superdex 75 prep grade, can also be Sephacryl S-200 High Resolution, working concentration be that the phosphate buffered saline buffer wash-out of 5～50mmol/L, pH value 7.0～8.5 obtains target protein CNTF.
Embodiment
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described by preferred embodiment.But should be appreciated that these embodiment only are for the present invention being described, protection scope of the present invention not being had any restriction.Essence of the present invention and protection domain are only limited by additional claims.
Embodiment 1: the genetic engineering bacterium that obtains to express the rhCNTF mutant
We are cloned into natural CNTF gene order by the RT-PCR reaction from the human blood cell, and obtain following rhCNTF mutant sequence through sudden change:
SEQ ID NO.1：
catatggctt tcacagagca ttcaccgctg acccctcacc gtcgggacct
cgcaagccgc tctatctggc tagcaaggaa gattcgttca gacctgactg
ctcttacgga atcctatgtg aagcatcagg gcctgaacaa gaacatcaac
ctggactctg cggatgggat gccagtggca agcactgatc gttggagtga
gctgaccgag gcagagcgac tccaagagaa ccttcaagct tatcgtacct
tccatgtttt gttggccagg ctcttagaag accagcaggt gcattttacc
ccaaccgaag gtgacttcca tcaagctata catacccttc ttctccaagt
cgctgccttt gcataccaga tagaggagtt aatgatactc ctggaataca
agatcccccg caatgaggct gatgggatgc ctattaatgt tggagatggt
ggtctctttg agaagaagct gtggggccta aaggtgctgc aggagctttc
acagtggaca gtaaggtcca tccatgacct tcgtttcatt tcttctcatc
agactgggta ataa
ggatcc
Underscore part wherein
CatatgWith
GgatccBe followed successively by NdeI and BamHI restriction enzyme site, dash area
AtgBe followed successively by the initiator codon and the terminator codon of genetic expression with taataa.This mutant sequence is the preferably combination of aforementioned mutation method, promptly compare with natural hCNTF gene, the halfcystine that is the 17th replaces with L-Ala, the 63rd L-glutamic acid replaces with arginine and removes 15 amino acid whose sudden change combinations of C-terminal (rhCNTF-TS), the NdeI (691) and BamHI (198) site (referring to the Novagen company plasmid map) that utilize NdeI on this sequence and BamHI restriction endonuclease sites to be cloned into pET32a (+) prokaryotic expression plasmid, obtain the recombinant expression vector plasmid, with this vector plasmid transformed into escherichia coli BL21 (DE3) competence, obtain to express the engineering bacteria of rhCNTF mutant, mutant of expressing and natural hCNTF are relatively, the 17th halfcystine replaces with L-Ala, the 63rd L-glutamic acid replaces with arginine, and C-terminal is removed 15 amino acid.This project bacterium has carried out biological preservation on March 22nd, 2005 at China typical culture collection center (CCTCC, Wuhan University, postcode 430072), and deposit number is: CCTCC NO:M205025.
Embodiment 2:IPTG induces engineering bacterium expression rhCNTF mutant
Get the frozen recombination engineering bacteria BL21 (DE3) of glycerine pipe, the inoculum size by 0.2% inserts in the fresh LB resistance substratum and (contains Amp100 μ g/ml), incubated overnight.Activatory kind liquid is transferred in the 500ml triangular flask that 200ml LB substratum is housed by 1% inoculum size, 280rpm, 37 ℃ are cultured to A
600During=0.6 left and right sides, be that the IPTG of 0.5mmol/L induces with the final concentration, continue again to cultivate 4 hours, the solubility expression of the formation of the target protein 90% or more as a result, the soluble-expression amount is 20～25%.(Fig. 1).
Embodiment 3: lactose-induced rhCNTF mutant is at the efficient soluble-expression that shakes under bottle condition
Get the frozen genetic engineering bacterium BL21 (DE3) of glycerine pipe, the inoculum size by 0.2% inserts in the fresh LB resistance substratum and (contains Amp100 μ g/ml), incubated overnight.Activatory kind liquid is transferred in the 500ml triangular flask that the 200mlLB substratum is housed by 1% inoculum size, 280rpm, 37 ℃ are cultured to A
600During=0.6 left and right sides, the adding final concentration is 2% lactose solution, 220rpm, and 25 ℃ are continued to cultivate 3000g centrifugal force, 10 minutes centrifugal results thalline 6 hours.Weigh wet thallus weight, with the Tris-HCl damping fluid of 50mmol/L in the resuspended thalline of 1: 20 ratio.Behind the broken bacterium of ultrasonic wave, broken bacterium liquid is with 20,000g centrifugal force, 10 minutes centrifugal, takes out supernatant, precipitates resuspended with isopyknic damping fluid, to break bacterium liquid supernatant respectively, the parallel SDS-PAGE that carries out of the thalline of precipitation and results, the target protein more than 90% forms solubility expression as a result, and the soluble-expression amount is about 20%.Fig. 2 is the expression of the rhCNTF mutant in each period after inducing.
Embodiment 4: the efficient soluble-expression of lactose-induced rhCNTF mutant under fermentor tank amplification culture condition
Get the frozen genetic engineering bacterium BL21 (DE3) of glycerine pipe, streak inoculation LB resistance inclined-plane is cultivated to take out after 12～18 hours for 37 ℃ and is put 4 ℃ of preservations.A slant strains is changed in the triangular flask that the 400ml seed culture fluid is housed, 280rpm, 37 ℃ are cultured to A
600About=1.5.Seed culture fluid is the LB substratum.
In the 14L automatic fermenter, the 7L fermention medium of packing into.Fermention medium consists of: Tryptone 10g/L, Yeast Extract 5g/L, NaCl 10g/L, glucose 2g/L, casein hydrolysate 2g/L, Na
2HPO
412H
2O14g/L, KH
2PO
44g/L, (NH
4)
2HPO
42g/L, lactose 0.9g/L.Wherein Tryptones and yeast extract are the OXOID product, and casein hydrolysate is homemade biochemical reagents, and all the other are homemade analytical reagent.Substratum is with 121 ℃, and real jar was sterilized in 25 minutes.Sterilization finish treat substratum cooling after, insert 400ml cultured seed liquid, control pH6.8, oxygen dissolving value 40%, 37 ℃ of temperature, oxygen dissolving value is by mixing speed and air flow cascade.3.5 after hour, stream adds additional liquid of carbon source and the additional liquid of nitrogenous source simultaneously in fermentor tank, the flow acceleration by additional liquid of in good time adjustment carbon source and the additional liquid of nitrogenous source makes the bacterium specific growth rate be controlled at 0.2h
-1About.Wherein the additional liquid of carbon source consists of: glucose 200g/L, MgSO
47H
2O 10g/L, lactose 8g/L; Nitrogenous source replenishes liquid and consists of: Tryptones 40g/L, yeast extract 20g/L, casein hydrolysate 10g/L, MgSO
47H
2O 10g/L, lactose 8g/L.
Be cultured to A
600=12～14 o'clock, the lactose solution of disposable adding 30% in fermentor tank, making its final concentration is 1.7%, and the stream that suspends the additional liquid of carbon source this moment adds, and adjusting oxygen dissolving value is 20%.After 2～2.5 hours, recover stream and add the additional liquid of carbon source, control bacterium specific growth rate 0.05～0.15h
-1, adjust 25～28 ℃ of culture temperature, oxygen dissolving value 10～15%.Continue to cultivate after 6 hours and put jar, the bacterium final concentration reaches A
600About=25, wet thallus output 50g/L.With the ratio of wet thallus,, under the pressure of 800bar, break 2 circulations of bacterium with high pressure homogenizer with the resuspended thalline of Tris-HCl damping fluid of 50mmol/L in 1: 10.Get brokenly bacterium liquid sample, 20000g centrifugal force, 10 minutes are centrifugal, take out supernatant liquor, precipitates resuspendedly with isopyknic damping fluid, will break bacterium liquid supernatant respectively, the thalline one of precipitation and the results SDS-PAGE that walks abreast.Target protein more than 90% forms solubility expression as a result, and the soluble-expression amount is about 20%.Fig. 3 is the expression of the rhCNTF mutant in each period after inducing.
The separation and the purifying of embodiment 5:rhCNTF mutant
With the wet thallus of fermentation results ratio,, under the pressure of 800bar, break 2 circulations of bacterium with high pressure homogenizer with the resuspended thalline of Tris-HCl pH8.0 damping fluid of 50mmol/L by 1: 10.To break bacterium liquid earlier and add ammonium sulfate to 20% saturation ratio, 10, the centrifugal 40min of 000g takes out supernatant, continues to add ammonium sulfate to 45% saturation ratio in 20% supernatant, and the rhCNTF precipitation is separated out, and 10, the centrifugal 25min of 000g abandons supernatant, reclaims precipitation.The resuspended precipitation of Tris-HCl pH8.0 damping fluid with the 50mmol/L that contains 0.6mol/L ammonium sulfate, centrifugal or behind 0.45 μ m filtering with microporous membrane, go up the XK50/30 Sepharose Butyl 4Fast Flow post of having got well with the Tris-HCl pH8.0 damping fluid balance of the 50mmol/L that contains 0.6mol/L ammonium sulfate with flow velocity 10ml/min, wear to the 280nm absorption curve steadily approaching with the Tris-HCl pH8.0 damping fluid wash of the 50mmol/L that contains 0.6mol/L ammonium sulfate earlier, use about 1.5 column volumes of Tris-HCl pH8.0 buffer solution elution of the 50mmol/L that contains 0.2mol/L ammonium sulfate again, an assorted peak appears, use the Tris-HCl pH8.0 buffer solution elution of 20mmol/L then, collect elution peak, this is a target peak, NaCl with proper amount of deionized water or 1mol/L regulates specific conductivity to 0.35～0.4ms/cm, go up the XK26/20 Sepharose Q Fast Flow post of having got well with the Tris-HCl pH8.0 damping fluid balance of 50mmol/L with flow velocity 10ml/min, use about 1.5 column volumes of Tris-HCl pH8.0 buffer solution elution of the 50mmol/L that contains 0.04mol/L NaCl earlier, an assorted peak appears, use the Tris-HCl pH8.0 buffer solution elution of the 50mmol/L that contains 0.14mol/L NaCl again, collect elution peak, this is a target peak, the ice bath magnetic agitation adds solid ammonium sulfate to 60% saturation ratio, centrifugal collecting precipitation.PB pH8.0 damping fluid with a small amount of 20mmol/L is resuspended, go up XK26/100 Superdex 75 posts of having got well through the PB of 20mmol/L pH8.0 damping fluid balance with flow velocity 2ml/min, PB pH8.0 buffer solution elution with 20mmol/L, collect active peak, this is the pure product of rhCNTF mutant, through the SDS-PAGE electrophoretic analysis, purity is greater than 98%, and it is 2.7mg/ml that its concentration is decided the protein method detection with lowry.
Embodiment 6: the activity of recombinant C NTF mutant detects
The method that recombinant C NTF mutant activity detects in the present embodiment is submitted in the application referring to the applicant preferential day, exercise question is the Chinese patent application No.200510066194.6 of " method of quantitative assay correlation factors of ciliary nerves trophic factor ", and it incorporates this paper by reference into.
In brief, the rhCNTF mutant protein sample that obtains in high sugared DMEM substratum dilution the foregoing description 5 with the penicillin that contains 100IU/ml, 100 μ g/ml Streptomycin sulphates and 4mmol/L glutamine, maximum concentration is 3000 times of dilutions, below use 9000 times of dilutions, 27000 times of dilutions etc. to make 3 times of gradient dilutions, totally 10 dilute samples.Obtain the chick embryonic dorsal root ganglion cell as the method in the above-mentioned patent application.High sugared DMEM substratum dilution neurocyte liquid to 50000 cell/ml with containing 2% serum joins in the 96 porocyte culture plates of mouse tail glue primordial covering every hole 100 microlitres.Treat that neurocyte is inhaled after adherent 2 hours and abandon the substratum that contains serum, every hole adds high sugared DMEM substratum 100 microlitres that contain different concns CNTF, each extent of dilution is done 3 repeating holes, get high sugared DMEM substratum that 3 holes add the same preparation do not contain sample in addition as negative control, be positioned in the incubator of 37 ℃ of temperature, gas concentration lwevel 5%, saturated steam and cultivated 48 hours.Exhaust nutrient solution in each hole successively, discard.Every hole adds 200 microlitre PBS damping fluid (0.058mol/L Na
2HPO
4, 0.017mol/L NaH
2PO
4, 0.068mol/LNaCl pH7.4), leaves standstill after 1 minute and exhausts PBS in each hole successively, discards.Add successively in each hole 100 microlitre 1mg/ml pNPP substrate solutions (take by weighing a certain amount of pNPP, add in proportion and comprise 0.1mol/L sodium acetate pH6.0,0.2% Triton X-100, the damping fluid of 1mmol/L EDTA dissolves it fully.Face and use preceding preparation, use in 1 hour), put 37 ℃.After treating 4 hours, every hole adds 10 microlitre 1mol/L NaOH, mixes.Room temperature was placed after 10 minutes, read absorbance in BIO-TEK microplate reader 405nm place, and is as shown in the table.According to the data work dose-effect curve as shown in Figure 6 that obtains, determine the median effective dose ED of sample
50, and definite activity is 1.63 * 10
7Unit/ml, specific activity are 6.05 * 10
6Unit/mg.
| Extension rate | 3000 | 9000 | 27000 | 81000 | 243000 | 729000 | 2187000 | 6561000 | 19683000 | 59049000 | Negative |
| Repeating hole | |||||||||||
| 1 repeating | 1.477 1.667 1.581 1.575 | 1.633 1.658 1.563 1.618 | 1.545 1.56 1.534 1.546 | 1.643 1.742 1.71 1.698 | 1.579 1.898 1.549 1.675 | 1.411 1.371 1.308 1.363 | 1.189 1.117 1.158 1.155 | 0.602 1.114 0.949 0.888 | 0.713 0.779 0.856 0.782 | 0.822 0.746 0.886 0.824 | 0.845 0.871 0.69 0.802 |
In sum, the recombinant C NTF mutant protein that is obtained by above-mentioned fermentation and separation purifying technique can promote external former chick embryonic dorsal root ganglion cell survival of being commissioned to train foster, and has tangible dose-effect relationship.
Claims (19)
1. a method for preparing the rhCNTF mutant comprises the following steps:
(a) rhCNTF mutant gene sequence clone is gone into expression vector obtaining recombinant vectors, and import in the intestinal bacteria, generate the genetic engineering bacterium that contains recombinant vectors;
(b) the culturing gene engineering bacteria is to A
600=12～14 o'clock, add lactose, induce rhCNTF mutant solubility expression in genetic engineering bacterium;
(c) induce after, in nutrient solution, slowly add glucose as main carbon source, and dissolved oxygen value is 10～15% in the control nutrient solution, continues to cultivate;
(d) treat the A of genetic engineering bacterium
600=20～30 o'clock, gather in the crops thalline, and bacterial cell disruption is obtained brokenly bacterium liquid;
(e) the broken bacterium liquid of purifying obtains purified product.
2. according to the process of claim 1 wherein that the sudden change that relates in the described rhCNTF mutant is selected from: the halfcystine that rhCNTF is the 17th replaces with L-Ala or arginine; The 63rd L-glutamic acid replaces with arginine; The 64th Histidine replaces with L-Ala; Remove 1～30 amino acid of C-terminal of rhCNTF; Or the combination of these sudden changes.
3. according to the method for claim 2, wherein said C-terminal amino acid is removed 13 or 15.
4. according to the method for claim 2 or 3, wherein said sudden change is combined as that the 17th halfcystine replaces with L-Ala, the 63rd L-glutamic acid replaces with arginine and C-terminal is removed 15 amino acid whose combinations.
5. according to the process of claim 1 wherein that the process of inducing of step (b) carried out 1～6 hour.
6. add lactose in the initial medium, in the additional liquid of carbon source, add lactose and in the additional liquid of nitrogenous source, add lactose according to the process of claim 1 wherein that the middle adding of step (b) lactose is included in.
7. according to the method for claim 6, the final concentration that wherein adds lactose is 1 weight %～2 weight %.
8. according to the method for claim 7, the final concentration that wherein adds lactose is 1.7 weight %.
9. according to the process of claim 1 wherein that the middle control of step (c) culture temperature is 25～28 ℃.
10. according to the process of claim 1 wherein the A of genetic engineering bacterium in the step (d)
600=25.
11. broken bacterium liquid is carried out saturation ratio 20%～45% grade ammonium sulfate salting-out, hydrophobic chromatography, anion-exchange chromatography and gel-filtration purifying successively according to the process of claim 1 wherein in the step (e), obtains purified product.
12. method according to claim 11, wherein said hydrophobic chromatography filler is Butyl Sepharose 4 Fast Flow or Phenyl Sepharose 4 Fast Flow, in the chromatography process, use the damping fluid balance chromatography column that contains ammonium sulfate, behind the last sample, with the assorted peak of the buffer solution elution of sulfur acid ammonium, use the not active peak of buffer solution elution of sulfur acid ammonium more earlier.
13. according to the method for claim 12, the ammonium sulfate concentrations scope that level pad wherein contains is 0.5～1.0mol/L; Washing the ammonium sulfate concentrations scope that assorted peak damping fluid contains is 0.2～0.3mol/L; Damping fluid is Tris-HCl, and concentration range is 10～50mmol/L, and the pH value is 7.0～9.0.
14. method according to claim 11, anion-exchange chromatography filler wherein is Q Sepharose Fast Flow or DEAE Sepharose Fast Flow, use the damping fluid balance chromatography column that contains salt in the chromatography process, behind the last sample, earlier with the assorted peak of saliniferous buffer solution elution, again with containing the more active peak of buffer solution elution of high salt concentration.
15. according to the method for claim 14, the damping fluid of use is the Tris-HCl damping fluid, concentration is 20～50mmol/L, and the pH value is 7.5～8.5.
16. method according to claim 14 or 15, contained salt is sodium-chlor in the damping fluid, the sodium chloride concentration that contains in the damping fluid at the assorted peak of the first step wash-out is 0.02～0.05mol/L, and the sodium chloride concentration that contains in the damping fluid at the active peak of the second step wash-out is 0.13～0.20mol/L.
17. according to the method for claim 11, it also comprises the step that concentrates activated protein solution by ammonium sulfate precipitation between anion-exchange chromatography and gel permeation chromatography step.
18. method according to claim 11, the filler of described gel permeation chromatography is Superdex 75 prep grade or Sephacryl S-200 High Resolution, use phosphate buffered saline buffer to carry out balance and wash-out, concentration is 5～50mmol/L, and the pH value is 7.0～8.5.
19. according to the purity of any described method preparation in the claim 1～18 greater than 98%, and the rhCNTF mutant of biologically active.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2006100767199A CN100549174C (en) | 2005-04-21 | 2006-04-18 | Mutant of recombined human ciliary nerves nutrilitc and preparation method thereof |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510066193 CN1687413A (en) | 2005-04-21 | 2005-04-21 | Mutant of recombined human ciliary nerves nutrilitc and preparing method thereof |
| CN200510066193.1 | 2005-04-21 | ||
| CNB2006100767199A CN100549174C (en) | 2005-04-21 | 2006-04-18 | Mutant of recombined human ciliary nerves nutrilitc and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1844392A true CN1844392A (en) | 2006-10-11 |
| CN100549174C CN100549174C (en) | 2009-10-14 |
Family
ID=37063362
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2006100767199A Expired - Fee Related CN100549174C (en) | 2005-04-21 | 2006-04-18 | Mutant of recombined human ciliary nerves nutrilitc and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100549174C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305522A (en) * | 2013-06-20 | 2013-09-18 | 江苏省原子医学研究所 | Optimized and recombined nucleotide sequence of human ciliary neurotrophic factor and efficient soluble expression method in escherichia coli |
| CN103800894A (en) * | 2014-03-03 | 2014-05-21 | 山东省眼科研究所 | Application of CNTF to corneal limbal stem cell proliferation and corneal epithelium damage repair |
| CN104193817A (en) * | 2014-09-05 | 2014-12-10 | 桂林英美特生物技术有限公司 | Process for purifying human retinol binding protein and preparation process of polyclonal antibody thereof |
| CN104558148A (en) * | 2013-10-17 | 2015-04-29 | 北京生物制品研究所有限责任公司 | Ciliary neurotrophic factor mutant, and modified mutant and application thereof |
-
2006
- 2006-04-18 CN CNB2006100767199A patent/CN100549174C/en not_active Expired - Fee Related
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103305522A (en) * | 2013-06-20 | 2013-09-18 | 江苏省原子医学研究所 | Optimized and recombined nucleotide sequence of human ciliary neurotrophic factor and efficient soluble expression method in escherichia coli |
| CN103305522B (en) * | 2013-06-20 | 2015-11-25 | 江苏省原子医学研究所 | A kind of nucleotide sequence of optimum combination Human Ciliary Neurotrophic Factor and in intestinal bacteria solution expression with high efficiency method |
| CN104558148A (en) * | 2013-10-17 | 2015-04-29 | 北京生物制品研究所有限责任公司 | Ciliary neurotrophic factor mutant, and modified mutant and application thereof |
| CN103800894A (en) * | 2014-03-03 | 2014-05-21 | 山东省眼科研究所 | Application of CNTF to corneal limbal stem cell proliferation and corneal epithelium damage repair |
| CN103800894B (en) * | 2014-03-03 | 2015-04-29 | 山东省眼科研究所 | Application of CNTF to corneal limbal stem cell proliferation and corneal epithelium damage repair |
| CN104193817A (en) * | 2014-09-05 | 2014-12-10 | 桂林英美特生物技术有限公司 | Process for purifying human retinol binding protein and preparation process of polyclonal antibody thereof |
| CN104193817B (en) * | 2014-09-05 | 2016-09-14 | 桂林英美特生物技术有限公司 | The purifying process of human retinol-binding protein and the preparation technology of polyclonal antibody thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100549174C (en) | 2009-10-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1687413A (en) | Mutant of recombined human ciliary nerves nutrilitc and preparing method thereof | |
| CN107893087A (en) | A kind of preparation method and applications of the interferon of Recombinant Swine λ 3 | |
| CN103467584B (en) | The acquisition of a kind of prokaryotic gene engineering heterozygosis cationic antibacterial peptide CC and fermentation process thereof | |
| CN100549174C (en) | Mutant of recombined human ciliary nerves nutrilitc and preparation method thereof | |
| Wake et al. | Extracts of marine cyanobacteria stimulated somatic embryogenesis of Daucus carota L. | |
| CN105461799A (en) | Purifying method for human recombinant follicle-stimulating hormone | |
| CN1757723A (en) | Method of producing recombination human bone morphopoiesis protein | |
| CN105368806A (en) | Site-directed mutagenesis modified saccharomyces cerevisiae dipeptidyl peptidase III | |
| CN1284797C (en) | Method for constructing, expressing and purifying human recombination factor and application | |
| CN101555485A (en) | Non-fusion expression of recombinant human parathyroid hormone (1-84) and large-scale preparation method | |
| CN1233839C (en) | Productive method of recombinant human liver regenerated enhanced factor and use in treating serious hepatopathy thereof | |
| CN102586257A (en) | Sika deer IGF (Insulin-like Growth Factor)-1 polypeptide and preparation method thereof | |
| CN1212336C (en) | Prepn of recombinant human parathyroid hormone PTH (1-34) | |
| CN1059237C (en) | Method for producing recombined human liver cytokinin and its use for curing serious hepatic disease | |
| CN101089181A (en) | Production method of recombination human interleukin-4 | |
| CN1112934C (en) | Epidemic hemorrhagic fever passage cell polyvalent purified vaccine | |
| CN1158388C (en) | Technological process of continuously perfused culture of hybrid tumor and CHO cell to prepare antibody | |
| CN105755008A (en) | DNA molecule for coding porcine alpha interferon and expression and purification method of recombinant protein of porcine alpha interferon | |
| CN109402095A (en) | A kind of protease and its method for preparing the low big mpd polypeptide of cadmium | |
| CN1840682A (en) | Mass production of high activity thymosin al by genetic engineering method | |
| CN100523172C (en) | Gene engineering bacteria of high efficiency expression of human alpha 1-thymulin and its construction method and use | |
| CN1896096A (en) | Production of high-activity extrasin beta 4 derivative by gene engineering | |
| CN106498013A (en) | The industrialization preparation method of recombinantinterferonα 1 and application | |
| CN1259337C (en) | Synthesis, expression, preparation and application for human parathyroid hormone gene mutant | |
| CN101260143A (en) | Highly effective production method for canine leucocyte interferon |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20091014 Termination date: 20190418 |