CN101144082B - Recombination human ciliary neurotrophy factor, mutant and application thereof - Google Patents
Recombination human ciliary neurotrophy factor, mutant and application thereof Download PDFInfo
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Abstract
The present invention relates to a recombinant human ciliary nerve nutrition factor, a mutant of the nutrition factor, and the preparation and the application thereof. The present invention provides the recombinant human ciliary nerve nutrition factor that the 17th thioserine of a natural CNTF gene is changed into alanine, the 63rd glutamic acid of the protogene is changed into arginine, at the same time, the partial codons in the CNTF protogene are changed into the codons which is favored by prokaryotes, but the code of the gene is not changed. On the basis, the recombinant CNTF is modified, to form different mutants. The mutants of the present invention has better effect on the aspect of curing the ordinary obesity or diabetes type obesity.
Description
Technical field
The present invention relates to recombination human ciliary neurotrophy factor, the two mutants of this nutritional factor, and their preparation and application.
Background technology
CNTF (CNTF) is the neurocyte factor, begins from late embryogenesis, and expression is all arranged in periphery and the cns, and its molecular weight is 22.75KDa, comprises 200 amino acid.Existing document discloses and adopts the Human Ciliary Neurotrophic Factor (CNTF) of recombination to be used to treat common bariatric and mellitus type obesity.As: Chinese invention patent 200410030756 is openly used CNTF and derivatives for treatment obesity thereof; Other molecule that Chinese invention patent 97180757 discloses hCNTF and two mutants thereof or activated the CNTF acceptor is used for the purposes on the medicine of treatment of obesity and relative disease (for example hyperglycemia) in preparation.But natural Ciliary Neurotrophic Factor Gene is directly carried out expression efficiency in intestinal bacteria lower, and the stability of expressing protein is relatively poor relatively.For addressing these problems the modes that Ciliary Neurotrophic Factor Gene is suddenlyd change and modified that adopt more.
Summary of the invention
The present invention provides a kind of recombination human ciliary neurotrophy factor and this recombination human ciliary neurotrophy factor gene is changed structure and forms new two mutants.The present invention provides preparation of this two mutants and uses thereof simultaneously.
Recombination human ciliary neurotrophy factor of the present invention; Be that the 17th halfcystine with natural CNTF gene changes L-Ala into; The 63rd L-glutamic acid of protogene is changed to l-arginine; Simultaneously the codon in the original CNTF gene of part is changed into the codon of prokaryotic organism hobby, but do not change the coding (being that aminoacid sequence is constant) of gene.
Recombination human ciliary neurotrophy factor of the present invention, its recombination human ciliary neurotrophy factor gene order table is < 400>1.
The two mutants of first recombination human ciliary neurotrophy factor of the present invention is to remove 16 amino acid of the proteic C end of natural CNTF.
The two mutants of second recombination human ciliary neurotrophy factor of the present invention is that proteic the 183rd Stimulina of natural CNTF sported L-glutamic acid, and 184 Threonines sport L-glutamic acid, and removes 16 amino acid of C end.
The two mutants of the 3rd recombination human ciliary neurotrophy factor of the present invention is that proteic the 183rd Stimulina of natural CNTF sported Methionin, and 184 Threonines sport Methionin, and removes 16 amino acid of C end.
The two mutants of the 4th recombination human ciliary neurotrophy factor of the present invention is to be Methionin with proteic the 185th glycine mutation of natural CNTF, and 186 isoleucine mutations are methionine(Met), removes 14 amino acid of C end.
The two mutants of the 5th recombination human ciliary neurotrophy factor of the present invention is a halfcystine with the 17th alanine mutation of natural CNTF albumen; With the 185th glycine mutation is Methionin; 186 isoleucine mutations are methionine(Met), remove 14 amino acid of C end.
The two mutants of the 6th recombination human ciliary neurotrophy factor of the present invention is to be Serine with the 17th alanine mutation, is Methionin with the 185th glycine mutation, and 186 isoleucine mutations are methionine(Met), removes 14 amino acid of C end simultaneously.
The two mutants of the 7th recombination human ciliary neurotrophy factor of the present invention is to remove natural CNTF albumen n end the 2nd～11 amino acids; With the 185th glycine mutation is Methionin; 186 isoleucine mutations are methionine(Met), remove 14 amino acid of C end simultaneously.
The preparation method of the two mutants of recombination human ciliary neurotrophy factor of the present invention is: change the 17th halfcystine of natural CNTF gene into L-Ala; The 63rd L-glutamic acid of protogene is changed to l-arginine, changes the codon in the original CNTF gene of part into obtain behind the codon of prokaryotic organism hobby recombinant C NTF simultaneously.CNTF with reorganization is a template again, with the purpose two mutants of aforesaid arbitrary recombination human ciliary neurotrophy factor for title product designs corresponding primer, carry out polymerase chain reaction; Again amplification is obtained goal gene and inserts and to have among the expression vector pETr of T7 promotor, through order-checking and enzyme cut identify correctly after, carry out protein induced expression; Expression product inserted in the substratum cultivate, add isopropyl-(IPTG) and carry out abduction delivering, the centrifugal collection thalline of 5000 * g; Add damping fluid, process the thalline suspension, the ultrasonication thalline; Centrifugal results inclusion body; This inclusion body with the dissolving of the damping fluid of hydrochloric guanidine or urea, is obtained solubilization of inclusion bodies liquid, through dilution refolding or post refolding method with the inclusion body protein renaturation.Obtain CNTF protein renaturation liquid; CNTF protein renaturation liquid is carried out chromatography purification with anion-exchange column; Target protein with purifying carries out polishing purification through hydrophobic chromatography post or gel chromatography column again; Collect protein peak, after the filter membrane Sterile Filtration, obtain the two mutants purified product of needed recombination human ciliary neurotrophy factor.
Resulting recombination human ciliary neurotrophy factor of the present invention or two mutants can be used for treating common bariatric or mellitus type obesity.
The recombination human ciliary neurotrophy factor that changes structure through the present invention can efficiently express in intestinal bacteria, can increase the stability of expressing protein simultaneously, keeps or strengthen the biological activity of expressing protein.Maximum characteristics of the present invention are that mutant protein is bioactive also can effectively to reduce its immunogenicity simultaneously increasing, and have overcome prior art when the treatment that is used for obesity, the higher problem that influences medicine (treatments) effect of its immunogenicity.The present invention reduces the disulfide linkage in the target protein, so that the purifying of target protein and renaturation through changing the terminal hydrophilic and hydrophobic of expressing protein.
Description of drawings
Fig. 1 is the electrophorogram that recombinant human CNTF of the present invention and each two mutants thereof are expressed bacterium bacterial cell disruption product, and wherein swimming lane 1 is that original CNTF expresses bacterium, and 2～8 swimming lanes are respectively two mutants CN2～CN8 and express bacterium, and the 9th swimming lane is the molecular weight of albumen standard.After Fig. 2 was CNTF and each two mutants administration, each organized mouse mean body weight change curve.Fig. 3: after the CNTF administration, more above the average age for marriage Kunming mouse loss of weight result relatively.Fig. 4: after the CNTF long term administration, mouse changes of weight curve.Fig. 5: various dose, different structure CNTF administration are after one month, and mouse produces the antibody titers result.
Embodiment
Embodiment of the present invention below is provided:
The full gene of recombinant C NTF of the present invention is synthetic to be accomplished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd, carrying out gene when synthetic, requires to have designed NdeI and two restriction enzyme sites of AvrII respectively at the upper and lower ends of goal gene.The synthetic goal gene total length 603bp of institute in the cloning vector PUC18 plasmid of packing into, shears the correct goal gene of order-checking get off, and inserts among the expression vector pETr, carries out protein expression and order-checking.
Compare with natural people CNTF gene; The synthetic cDNA of institute of the present invention has changed the halfcystine of the 17th of Ciliary Neurotrophic Factor Gene into L-Ala; The 63rd L-glutamic acid has changed l-arginine into, simultaneously, partial password in the original CNTF gene is changed into the codon of prokaryotic organism hobby; But do not relate to amino acid whose change, belong to same sense mutation.Made up total length CNTF protein expression vector CN1 on this basis; That is: with the plasmid that contains the CNTF full-length gene of synthetic with NdeI and AvrII double digestion; Enzyme is cut product and is packed into and insert the corresponding position use the expression plasmid PETx that handles with enzyme, carries out protein expression.The CNTF PROTEIN C N1 of reorganization can efficiently express in intestinal bacteria, and it is proteic more than 35% that expression amount accounts for whole cell, and expressing protein has the proteic biological activity of natural CNTF and the drug effect that loses weight.
Structure at his recombinant C NTF two mutants of the enterprising Xingqi of last working foundation.
The sudden change that the present invention carried out and change structure and carry out from the following aspects has made up 7 two mutants altogether, and each two mutants is respectively CN2, CN3, CN4, CN5, CN6, CN7 and CN8.The primer that each two mutants is used and the change of structure are following:
1, the structure of CN2: the CN1 gene with foregoing synthetic is a template, synthetic upstream and downstream primer, and used upstream primer is:
5 '-CGATTGACCATATGGCTTTCACTGAACACTCTC-3 ', introduce the NdeI restriction enzyme site; Used downstream primer is: 5 '-CTACCACCTAGGTATTAAGTCTGATGAGAAGAGATG-3 ', introduce the AvrII restriction enzyme site, through pcr amplification, remove 16 amino acid of the proteic C end of natural CNTF.
2, the structure of CN3: the CN1 gene with aforementioned synthetic is a template, synthetic upstream and downstream primer, the same CN2 of used upstream primer; Downstream primer is: 5 '-GCAGCCTAGGTATTATTCCTCATGAG-3 '; Introduce the AvrII restriction enzyme site,, proteic the 183rd Stimulina sported L-glutamic acid through pcr amplification; 184 Threonines sport L-glutamic acid, remove 16 amino acid of C end.
3, the structure of CN4: the CN1 gene with aforementioned synthetic is a template, synthetic upstream and downstream primer, the same CN2 of used upstream primer; Downstream primer is: 5 '-GCAGCCTAGGTATTACTTCTTATGAG-3 '; Introduce the AvrII restriction enzyme site,, proteic the 183rd Stimulina sported Methionin through pcr amplification; 184 Threonines sport Methionin, remove 16 amino acid of C end.
4, the structure of CN5: the CNTF gene with aforementioned synthetic is a template, synthetic upstream and downstream primer, the same CN2 of used upstream primer; Downstream primer is: 5 '-GTACCCTAGGATTACATCTTAGTCTGATGAGAAG-3 '; Introducing the AvrII restriction enzyme site, through pcr amplification, is Methionin with the 185th glycine mutation of albumen; 186 isoleucine mutations are methionine(Met), remove 14 amino acid of C end.
5, the structure of CN6: the CN1 gene with aforementioned synthetic is a template; Synthetic upstream and downstream primer; Used upstream primer is: 5 '-TTATTGCATATGGCTTTCACAGAGCATTCACCGCTGACCCCTCACCGTCGGGACCT CTGTAGC-3 ', introduce the NdeI restriction enzyme site; The same CN5 of used downstream primer through pcr amplification, is a halfcystine with the 17th alanine mutation, is Methionin with the 185th glycine mutation of albumen, and 186 isoleucine mutations are methionine(Met), removes 14 amino acid of C end simultaneously.
6, the structure of CN7: the CN1 gene with aforementioned synthetic is a template; Synthetic upstream and downstream primer; Used upstream primer is: 5 '-TTATTGCATATGGCTTTCACAGAGCATTCACCGCTGACCCCTCACCGTCGGGACCT CTCTAGC-3 ', introduce the NdeI restriction enzyme site; The same CN5 of downstream primer through pcr amplification, is a Serine with the 17th alanine mutation, is Methionin with the 185th glycine mutation of albumen, and 186 isoleucine mutations are methionine(Met), removes 14 amino acid of C end simultaneously.
7, the structure of CN8: the CN1 gene with aforesaid synthetic is a template, synthetic upstream and downstream primer, and upstream primer is: 5 '-ATAATCCATATGCGTCGGGACCTCTGTAG-3 ', introduce the NdeI restriction enzyme site; The same CN5 of downstream primer, through pcr amplification, disappearance is fallen N and is held the 2nd～11 amino acids, is Methionin with the 185th glycine mutation of albumen, and 186 isoleucine mutations are methionine(Met), remove 14 amino acid of C end simultaneously.
The gene order of CN1 of the present invention and each two mutants CN2 to CN8, and primary CNTF gene order is seen after and is attached sequence table.
The aminoacid sequence of CN1 of the present invention and each two mutants CN2 to CN8, and primary CNTF aminoacid sequence is seen after and is attached sequence table.
With the above goal gene that respectively increases, insert and to have among the expression vector pETr of T7 promotor, through order-checking and enzyme cut identify correctly after, carry out protein induced expression, expressed CNTF target protein content accounts for more than 35% of total tropina.CNTF and each two mutants expressing protein result's thereof electrophorogram is seen Fig. 1.
Express CNTF induction expression of protein, renaturation and purifying process in front on the working foundation again.Its concrete practice is following:
Fermentation culture
The fermention medium that the present invention adopted is a complex medium, its composition with consist of (g/L): peptone 20, yeast powder 10, sodium-chlor 5.With fermentation seed liquid; Be aforementioned CN1; And each two mutants: CN2, CN3, CN4, CN5, CN6, CN7 and CN8, the ratio with 1: 10～1: 40 (V/V) inserts in the fermention medium respectively, controls suitable fermentation culture temperature, pH value, dissolved oxygen (DO); Carry out fermentation culture, whenever survey OD at a distance from sampling in a hour
600Value, work as OD
600At 5～6 o'clock, add isopropyl-(IPTG) and carry out abduction delivering, the final concentration of IPTG is 0.1mmol/L, induces and finishes after 4 hours to cultivate, with the centrifugal collection thalline of nutrient solution 5000 * g.
Renaturation and purifying
With PB or the Tris-HC1 damping fluid dilution of wet thallus, process the thalline suspension, ultrasonication with pH6.0～7.5; 8000 * g～1200 * g spinning, the results inclusion body, the damping fluid dissolving inclusion body with hydrochloric guanidine or urea obtains solubilization of inclusion bodies liquid.Through dilution refolding or post refolding method with the inclusion body protein renaturation.Obtain CNTF protein renaturation liquid; Anion-exchange column on the CNTF protein renaturation liquid is carried out chromatography purification; Target protein with purifying carries out polishing purification through hydrophobic chromatography post or gel chromatography column again; Collect protein peak, after the filter membrane Sterile Filtration, obtain the two mutants purified product of needed recombination human ciliary neurotrophy factor.If aforesaid renaturation, purifying process condition are carried out preferably can obtaining best effect, its purity can reach 98%.
For ease of preservation, can the filter membrane Sterile Filtration of gained CNTF stoste with 0.22 μ m be added the protective material freeze-drying, be the CNTF finished product.
Can adopt adequate protective agent when preparing oral prepns with the CNTF finished product, like enteric coated capsule, inclusion compounds such as slow releasing capsule, micro-capsule in CNTF is wrapped in, are processed capsule formulation.
The weight loss effect of each two mutants expressing protein of CNTF of the present invention relatively adopts mouse loss of weight method, and concrete grammar is:
Select the cleaning level Kunming mouse of body weight at 16～20 grams, weigh in (being accurate to 0.1g), by the body weight random packet, every group of 10～20 animals.To establish standard substance or reference article control group and saline water or other solvent control groups simultaneously.Various recombinant C NTF expressing proteins are also prepared by the same procedure purifying; Every day, intraperitoneal administration twice, and administration is 8～10 days altogether, negative control group injecting normal saline or other solvents.Each organizes experimental animal in administration 0 day, weighs in once before administration every day.Each test group is given the mouse feed and the tap water of and q.s identical with composition every day.Observed altogether 10～12 days.Calculating body weight gain inhibiting rate (calculating formula is formula as follows)-be loss of weight efficient.
Recombinant C NTF of the present invention and each mutant protein are respectively organized mouse mean body weight result of variations after to administration and are seen table 1 and Fig. 2.
Respectively organize mouse each item every day index result after each mutant protein administration of table 1:CNTF
Test group dosage in the table: CNTF administration group, 10 mouse/groups, every mouse is calculated by kg body weight, every day dosage 100 micrograms, that is: 100 μ g/kgd; Control group: 10 mouse, per injection equal-volume saline water.Being tried mouse raising condition is: each test group is given the mouse feed and the tap water of and q.s identical with composition every day
Each organizes mouse mean body weight every day increment curve referring to Fig. 2.
Usually body weight is in the vigorous period of growing at the healthy kunming mice of 16～20 grams, about 1 gram of body weight gain every day.Can find out that from table 1 and Fig. 2 result the increment of mouse body weight obviously reduces after the CNTF administration.The above results shows that each sudden change group of CNTF is all obvious than the CNTF group mouse weight loss effect that do not suddenly change, and control group mice weight average after 10 days has increased by 15.7 grams; And each two mutants administration group mouse mean body weight of CNTF has only increased by 2.2～7.4 grams; All be lower than control group, the body weight gain inhibiting rate all is higher than 50%, wherein CN5 two mutants group after 10 days mean body weight increment have only 0.9 gram; The body weight gain inhibiting rate does not almost increase weight up to 94.3%.
Recombinant C NTF of the present invention and two mutants expressing protein thereof are seen table 2 and Fig. 3 to the influence of more above the average age for marriage Kunming mouse body weight.Mean body weight 32.71 grams, the Kunming mouse in mouse about eight weeks in age are adopted in this experiment, and administering mode is identical with aforementioned mouse loss of weight method with dosage, that is:
1) CN1: recombinant C NTF group, 10 mouse/groups, 100 μ g/kgd.
2) CN5:CNTF two mutants CN5 goods group, 10 mouse/groups, 100 μ g/kgd.
3) control group: 10 mouse/groups, inject equal-volume saline water every day.
Being tried mouse raising condition is: each test group is given the mouse feed and the tap water of and q.s identical with composition every day
The effect of CN5CN2, CN3, CN4, CN6, CN7, CN8 all is superior to CN1 in this group experiment, but is worse than the effect of CN5.Therefore in table 2 and Fig. 3, omit these experimental datas.
Table 2; CNTF to above the average age for marriage Kunming mouse administration after, changes of weight result
Can find out that from table 2 and Fig. 3 when the mouse body weight was higher, weight loss effect was also more obvious after the medication, be increased to 38.86 grams from 32.12 grams, have a net increase of long 6.75 grams like control group body weight after 9 days; And CN1 administration after 9 days body weight be reduced to 32.17 grams, loss of weight 0.84 gram from 33.01; Two mutants CN5 administration is after 9 days, and the mouse body weight becomes 28.17 grams from 33.00; Loss of weight 4.83 grams; The above results shows that the CNTF two mutants all has the loss of weight drug effect with the CNTF of not sudden change, but the proteic loss of weight drug effect of two mutants CNTF is more remarkable.
Recombinant C NTF of the present invention and the long term administration of two mutants expressing protein thereof; Drug effect and immunogenicity are relatively selected the healthy BALB/C mice of 16～18 grams for use; Successive administration 30 days (one month); After observing relatively various dose CNTF two mutants CN5 and CN1 injection mouse, the lose weight titre of situation and generation antibody of mouse.Mouse feed and the tap water of giving and q.s identical with composition every day respectively organized in experiment, and CNTF administration and dosage are following:
1) control group: 10 mouse, inject equal-volume saline water every day.
2) the CN1:CNTF product group of not suddenling change, 10 mouse, 100 μ g/kgd.
3) the CN1:CNTF product group of not suddenling change, 10 mouse, 200 μ g/kgd.
4) the CN1:CNTF product group of not suddenling change, 10 mouse, 600 μ g/kgd.
5) CN5:CNTF two mutants goods group, 10 mouse, 100 μ g/kgd.
6) CN5:CNTF two mutants goods group, 10 mouse, 200 μ g/kgd.
7) CN5:CNTF two mutants goods group, 10 mouse, 600 μ g/kgd.
Experimental result is seen table 3, Fig. 4 and Fig. 5.
Table 3CNTF administration is after one month, and each organizes the mouse antibodies titre:
The result can find out from table 3 and Fig. 4,5, and after long term administration, the antibody titers of CN5 is significantly less than CN1, and drug effect is superior to CN1, and this result becomes more obvious along with the prolongation of administration time.When detecting the immunogenicity of CNTF, select comparatively responsive BALB/C strain mouse for use, the characteristics of this strain mouse be responsive to immunogen, body weight is light and increases slowly.
Two mutants CN2, CN3, CN4, CN6, CN7, CN8 are experimentized, and its effect all is superior to CN1, but is worse than CN5, omits this group data at this
From above-mentioned experimental result, can draw to draw a conclusion:
CNTF two mutants of the present invention has tangible body weight gain to suppress or the drug effect that loses weight to experiment mice, and drug effect and dosage are tangible positive correlation effect (perhaps being dose-dependently).And the loss of weight drug effect of CNTF two mutants obviously is better than the CNTF that do not suddenly change.
In the two mutants group and the mouse loss of weight of the CNTF group of not suddenling change are tested; Through relatively finding out; Each two mutants group of CNTF is all obvious than the mouse weight loss effect of the CNTF group of not suddenling change, and wherein to suppress the weight of mice drug effect the most outstanding for CN5 two mutants group, in 100 μ g/kgd administrations after 10 days; The increment of mouse mean body weight has only 0.9 gram, and the body weight gain inhibiting rate is up to 94.3%.
In the loss of weight test of pesticide effectiveness of CNTF to above the average age for marriage Kunming mouse, when the mouse body weight was higher, weight loss effect was also more obvious after the medication,, decreased on the contrary in administration not only not increase of body weight after 9 days like CN1, was reduced to 32.17 grams, loss of weight 0.84 gram from 33.01; Two mutants CN5 administration is after 9 days, and the mouse body weight becomes 28.17 grams from 33.00; Loss of weight 4.83 grams; The above results shows that the CNTF two mutants all has the loss of weight drug effect with the CNTF of not sudden change, but the proteic loss of weight drug effect of two mutants CNTF is more remarkable.
In the CNTF long-term dosing study, can find out that the antibody generation time of CNTF two mutants CN5 is later than CN1, and antibody titers is significantly less than CN1, so its drug effect is superior to CN1; And this result is along with the prolongation of administration time becomes more obvious.CNTF two mutants experimental group is more obvious than the CNTF group mouse weight loss effect that do not suddenly change, and especially along with the prolongation of administration time, the CNTF that do not suddenly change group mouse body weight is gone up to some extent; CNTF two mutants group is then gone up not obvious, and this mainly is because of the prolongation along with administration time, begins to produce antibody in the mouse body; Because the antibody titers that CN1 produces is higher, (1:460～1:1140), a part of CNTF has therefore neutralized; Reduced drug effect, and along with the increasing of dosage, the antibody titers of generation increases also; Influence to drug effect is also more obvious, and the bounce-back of body weight is also more remarkable.(1:30～1:240), along with the increasing of dosage, though the antibody titers that produces also increases, drug effect is had certain influence under the enough big situation of dosage, still can keep drug effect but the CN5 group is owing to the antibody titers that produces is lower.The immunogenicity that the CNTF two mutants is described is lower than the not CNTF of sudden change.
Sequence table
< 110>Lanzhou Institute of Biological Products
< 120>recombination human ciliary neurotrophy factor, two mutants and application thereof
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Claims (2)
1. recombination human ciliary neurotrophy factor, it is characterized in that: change the 17th of natural Human Ciliary Neurotrophic Factor CNTF gene into L-Ala, the 63rd changes l-arginine into; Partial password in the gene changes the codon of prokaryotic organism hobby into; Proteic the 185th glycine mutation is Methionin, and 186 isoleucine mutations are methionine(Met), removes 14 amino acid of C end; Its gene order is SEQ ID NO:9, and its protein sequence is SEQ ID:10.
2. the application of the described recombination human ciliary neurotrophy factor of claim 1 in the medicine of preparation treatment common bariatric and mellitus type obesity.
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Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101555284B (en) * | 2008-04-10 | 2013-04-24 | 中国人民解放军军事医学科学院毒物药物研究所 | Truncated-type human ciliary nerve nutrition factor active segment and fusion protein thereof |
| 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 |
| CN103845727B (en) * | 2013-10-31 | 2016-01-20 | 上海现代药物制剂工程研究中心有限公司 | Nasal administration composition of recombination human ciliary neurotrophy factor and preparation method thereof |
| 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 |
| WO2016070419A1 (en) * | 2014-11-07 | 2016-05-12 | 陕西麦科奥特科技有限公司 | Ciliary neurotrophic factor nasal delivery system and preparation method and use thereof |
| CN104650215B (en) * | 2015-03-03 | 2018-02-13 | 上海医药工业研究院 | Human ciliary nerve trophic factor mutant, production method and purposes |
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| CN1387568A (en) * | 1999-08-13 | 2002-12-25 | 里珍纳龙药品有限公司 | Modified ciliary neurotrophic factor, method of making and methods of use thereof |
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2007
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| CN101144082A (en) | 2008-03-19 |
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