CN1752108A - Soluble tumour necrosis factor receptor II- - Google Patents
Soluble tumour necrosis factor receptor II- Download PDFInfo
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- CN1752108A CN1752108A CN 200510100468 CN200510100468A CN1752108A CN 1752108 A CN1752108 A CN 1752108A CN 200510100468 CN200510100468 CN 200510100468 CN 200510100468 A CN200510100468 A CN 200510100468A CN 1752108 A CN1752108 A CN 1752108A
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- gad
- stnfr
- fusion rotein
- adiponectin
- necrosis factor
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Abstract
A dual-functional soluble tumor necrosis factor receptor II-''lipo-linking substance'' globe fusion protein ( sTNFR II-gAD fusion protein) is prepared through reforming the cDNAs of sTNFR II and gAD to obtain sTNFR II-gAD fusion gene, configuring sTNFR II-gAD expression plasmid, and transferring it to colibacillus with high expression. It has the activity of both sTNFR II and gAD, that is, the antagonistic activity to TNF alpha and the ''lipo-splitting substance'' globe activity. It can be used for treating rheumatic arthritis, diabetes B, obesity, atherosclerosis and fatty liver.
Description
One, technical field
The invention belongs to the genetically engineered field, be soluble tumor necrosis factor receptor II-" adiponectin " bulb fusion rotein, i.e. sTNFR II-gAD.
Two, background technology
" adiponectin " is a kind of proteohormone of finding recently (Adiponectin), because of it is found by three different laboratories are successively independent, so claim GBP-28 again, apM1, AdipoQ and Acrp30; It to blood circulation, accounts for 0.01% (5-30 μ g/ml) of human plasma total protein, so its concentration is 10 of most of hormone concentrations by the synthetic justacrine of adipocyte
3-10
6Doubly (Shimada K, Miyazaki T, Daida H. (2004) Adiponectin and atherosclerotic disease.Clin Chim Acta.344:1-12.).Its protein structure is made up of two portions: the complement Clq sample ball-like structure of N-terminal collagen shape structure and carboxyl terminal.Adiponectin exists with two kinds of forms of the ball-like structure fragment (gAD) behind total length and the protease cracking in blood.Adiponectin can form homotrimer closely by its ball-like structure, then further form more complicated homology polymer by its collagen shape structure, but gAD only can form homotrimer (Berg AH, Combs TP, Scherer PE. (2002) Acrp30/adiponectin:an adipokine regulating glucose and lipid metabolism.TrendsEndocrinol Metab.13:84-89.).Adiponectin has two kinds of acceptor AdipoR1 and AdipoR2, AdipoR1 great expression in skeletal muscle wherein, and AdipoR2 is great expression in liver then; Total length adiponectin and AdipoR2 have higher affinity, the affinity that gAD is then higher with AdipoR1, although both all can combine with AdipoR1 and AdipoR2, and by activating AMP kinases and PPAR-α approach, promote Skeletal Muscle Cell and liver cell to glucose uptake and oxidation of fatty acids (Yamauchi T, Kamon J, Ito Y, et a1. (2003) Cloning of adiponectin receptors that mediate antidiabetic metabolic effects.Nature.423:762-769.).Experimentation on animals shows, it can promote the oxidation of body fat acid by muscle (account for weight of mammal 25%), reduces blood triglyceride, and by promoting the glucose metabolism of insulin sensitivity property improvement; The mouse of adiponectin disappearance is rendered as insulin resistant (insulin resistance), finally develop into type ii diabetes (Maeda N, Shimomura I, Kishida K, et al. (2002) Diet-induced insulin resistance in micelacking adiponectin/ACRP30.Nat Med.8:731-737.).It can be by suppressing monocyte/macrophage adhesion, migration and to the picked-up and the accumulation of modified low density lipoprotein, thereby suppress atherosclerotic formation.Clinical studies show, adiponectin concentration in obesity, type ii diabetes and the patients with coronary heart disease blood general low (Diez JJ, Iglesias P. (2003) The role of the novel adipocyte-derived hormone adiponectinin human disease.Eur J Endocrinol.148:293-300.).In addition, adiponectin can enter celiolymph by circulation and act on neuronal cell.The adiponectin that these central authorities discharge can make losing weight of mouse, and fat reduces; And it is effective too to lack the mouse that causes glucose level rising and fat to accumulate because of leptin (leptin).Be different from central leptin by reducing ingestion of food, central authorities' adiponectin then is by improving energy expenditure (the Qi Y that loses weight, Takahashi N, Hileman SM, et al. (2004) Adiponectin acts in the brain todecrease body weight.Nat Med.10:524-529.).Adiponectin can also be by promoting liver cell to oxidation of fatty acids with suppress the generation of the synthetic and TNF α of liver cell lipid acid, thereby slow down generation (the Xu A of alcohol/non-alcoholic fatty liver disease, Wang Y, Keshaw H, et al. (2003) The fat-derived hormone adiponectinalleviates alcoholic and nonalcoholic fatty liver diseases in mice.J Clin Invest.112:91-100.).Therefore, adiponectin is a cytokine with control type ii diabetes, obesity, atherosclerosis and fatty liver potentiality.
Soluble tumor necrosis factor receptor II (sTNFR II) is the natural agonist of TNF α, it is cytolemma outside part (the Mohler KM of Tumor Necrosis Factor Receptors II, Torrance DS, Smith CA, et al. (1993) Soluble tumor necrosis factor receptors are effective therapeutic agents in lethalendotoxemia and function simultaneously as both TNF carriers and TNF antagonists.JImmunol.151:1548-61.).The fused protein dimer (sTNFRII-Fc, called after Etanercept) that sTNFRII and IgG1 Fc (CH2 and CH3) form has been used for the treatment of rheumatoid arthritis from 1999 since the drugs approved by FDA clinically.Studies show that by the Dimerized effect of Fc, the ability of Etanercept anti-TNF alpha short of money is monomeric nearly 50 times of corresponding sTNFR II.Nearest numerous studies show that, TNF α also is the important virulence factor (Wisse BE. (2004) The inflammatory syndrome:The role of adipose tissue cytokines in metabolic disorderslinked to obesity.J Am Soc Nephrol 15:2792-2800.) in the pathologies such as type ii diabetes, obesity, atherosclerosis and fatty liver.Therefore, anti-TNF alpha short of money can not only be treated rheumatoid arthritis, and helps the control of type ii diabetes, obesity, atherosclerosis and fatty liver diseases.
It is highly important that, sTNFR II-gAD fusion rotein turns usefulness into by the tripolymer of gAD, impel sTNFRII to form homology two or tripolymer, can strengthen the avidity of this fusion rotein and TNF α greatly, and can prolong the transformation period of this fusion rotein in blood circulation because of molecular weight increases.In addition, combine with its acceptor AdipoR1 and AdipoR2 by gAD, the mixture that sTNFR II-gAD and TNF α interact and form can be directed to skeletal muscle and liver, finally TNF α can be removed from blood circulation.Therefore, no matter sTNFR II-gAD fusion rotein is on function, still on the ability of anti-TNF alpha short of money, the sTNFRII-Fc fusion rotein that all is better than present clinical use (promptly can only competitive inhibition TNF α, and it can not be removed from blood circulation, so must for a long time and continue medication).
Three, summary of the invention
The invention process of sTNFR II-gAD fusion rotein is as follows: in order to obtain to contain the plasmid of sTNFR II-gAD fusion gene, we have carried out clone and terminal the transformation to sTNFR II or gAD cDNA, have made up sTNFR II-gAD-pET24 recombinant plasmid; With this recombinant plasmid transformed e. coli bl21 (DE3), obtain corresponding engineering bacterium intestinal bacteria sTNFR II-gAD/pET24, and sTNFR II-gAD fusion gene has been realized expression, expression efficiency is 15-20%; Its expression product is respectively sTNFR II-gAD fusion rotein, has the bifunctional molecule activity, the activity in anti-TNF alpha promptly short of money and " fat splits element " bulb (comprising the oxidation, raising insulin sensitivity, inhibition atherosclerosis and the fatty liver that promote body fat acid).Fusion rotein mainly exists with the inclusion body form in bacterium; Separation and purification albumen and renaturation have obtained sTNFR II-gAD bifunctional fusion proteins after handling.6His is introduced the C end of fusion rotein, thereby be convenient to the separation and purification of this fusion rotein.
STNFR II-gAD bifunctional fusion proteins of the present invention wherein has the activity of anti-TNF alpha short of money (to hatch ED altogether with 0.25ng/mL TNF α
50=1-10ng/mL) and the biologic activity of adiponectin (as suppressing the propagation of mouse marrow leukaemia cell strain M1, half effectively suppresses dosage ED
50Be about 10 μ g/ml; And U-9889 (Streptozotocin) inductive type i diabetes mouse there is remarkable hypoglycemic effect.
STNFR II turns usefulness into by the tripolymer of gAD, impels sTNFR II to form homology two or tripolymer, thereby has strengthened the avidity of this fusion rotein and TNF α greatly, and can prolong the transformation period of this fusion rotein in blood circulation because of molecular weight increases.Combine with its acceptor AdipoR1 and AdipoR2 by gAD, the mixture that sTNFR II-gAD fusion rotein and TNF α interact and form can be directed to skeletal muscle and liver, finally TNF α can be removed from blood circulation.
Four, description of drawings
Fig. 1 is the synoptic diagram of plasmid sTNFR II-gAD-pET24.
Fig. 2 is sTNFR II-gAD fusion rotein efficiently expressing in intestinal bacteria.
Five, embodiment
By following embodiment technical characterictic of the present invention is described in detail.
Materials and methods:
One, animal, cell strain, bacterial strain and plasmid: mouse marrow leukaemia cell strain M1, inoblast strain L929, escherichia coli DH5a and BL21 (DE3); Prokaryotic expression plasmid pET24a (Kana
r, Novagen).
Two, main biochemical reagents and material: DNeasy organize the preparation test kit (Qiagen) of test kit and plasmid DNA, synthetic (Sigma) of oligonucleotide, Trizol, SuperScript II reversed transcriptive enzyme, Platinum Pfx archaeal dna polymerase and T4 dna ligase (Invitrogen); STNFR II-Fc (R ﹠amp; D Systems), agarose and SDS-PAGE (Biorad); Ni-NTA (Qiagen) and Sephacryl S-200 filler (Pharmacia).
Three, the connection of dna fragmentation, conversion and transformant screening, restriction endonuclease analysis, the equal reference literature of ordinary method (Sambrook J such as SDS-polyacrylamide gel electrophoresis, et al.Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 2nd edition, 1989) or the product description that provides of producer; Dna sequence analysis is finished in the dna sequencing service centre of UTSW.
Four, mouse type i diabetes Preparation of model: reference literature (Kunjathoor VV, Wilson DL, LeBoeufRC. (1996) Increased atherosclerosis in streptozotocin-induced diabetic mice.J ClinInvest 97:1767-1773.) with U-9889 (Streptozotocin, every day the 40mg/kg body weight, continuous 5 days, abdominal injection) induce male CL57BL/6 that type i diabetes (blood sugar concentration is 310-420mg/dl, and normal control is 160-230mg/dl) takes place.
The preparation of embodiment 1.gAD cDNA.
Play total RNA of human hypodermic fat tissue with the Trizol extracting, and with it as template, carry out RT-PCR and prepare gAD cDNA.
The preparation of embodiment 2.sTNFR II cDNA.
Play total RNA of peripheral blood mononuclear cell with the Trizol extracting, and with it as template, carry out RT-PCR and prepare sTNFR II cDNA.
Embodiment 3.sTNFR II-gAD-pET24 construction of recombinant plasmid.
Prepare sTNFR II-gAD-6His fusion gene with PCR, and carry out nucleotide sequencing; Then it is cloned respectively in pET24a, obtain sTNFR II-gAD-pET24 recombinant plasmid (Fig. 1), and limiting it property endonuclease analysis is identified.
Embodiment 4.sTNFR II-gAD fusion gene efficiently expressing in intestinal bacteria.
With sTNFR II-gAD-pET24 recombinant plasmid difference transformed into escherichia coli BL21 (DE3), obtain corresponding engineering bacterium sTNFR II-gAD/pET24; And sTNFR II-gAD fusion gene realized expression, and expression efficiency is 20-30%, mainly there be (Fig. 2) in expression product with the form of inclusion body.
The preparation of embodiment 5.sTNFR II-gAD-6His fusion rotein.
1. preparation inclusion body.5 gram thalline are suspended among the 100ml 1xPBS, ultrasonic in the ice bath (electric current 270mA), 30 seconds * 10 times (each 30 seconds at interval); Lysate is in 4 ℃ centrifugal 10 minutes (8000g), precipitation is suspended among the 100ml 1xPBS (including 4mol/L urea, 0.5%Triton X-100,20mmol/L EDTA) carries out rinsing, centrifugal subsequently (1000g * 10 minute) collecting precipitation; After twice of the rinsing, be dissolved in the 100mmol/L sodium phosphate buffer, in the 8mol/L urea (pH 8.0), 15000g * 15 minute supernatant.
2.Ni-NTA column chromatography: the solubilization of inclusion bodies liquid for preparing is splined on the Ni-NTA post, and (2.6 * 5cm), (100mmol/L sodium phosphate buffer, 8mol/L urea pH8.0) wash to sample A with balance liquid
280Return to baseline; (100mmol/L sodium phosphate buffer, 8mol/L urea pH4.5) carry out wash-out, collect fusion rotein mass peak (SDS-PAGE evaluation) to use elutriant then.
3. dialysis renaturation: the sTNFR II-gAD fusion rotein that the Ni-NTA column chromatography purification is obtained transfers to OD
280=0.2,4 ℃ of dialysis renaturation are 12 hours in greater than the dialyzate of 20 times of volumes (the 1mmol/L reduced glutathione, 0.2mmol/L oxidized form Triptide, pH 9.0 for 100mmol/L NaHCO3,1.0mol/L urea); At 50mmol/L NaHCO3,500mmol/L NaCl continues dialysis 6 hours among the pH 11 then; The centrifugal insolubles of removing is collected supernatant.
The active mensuration of embodiment 6.sTNFR II-gAD.
1. the functional examination of anti-TNF alpha short of money: reference literature (Mohler KM; Torrance DS; Smith CA; etal. (1993) Soluble tumor necrosis factor receptors are effective therapeutic agents inlethal endotoxemia and function simultaneously as both TNF carriers and TNFantagonists.J Immunol.151:1548-61.); under the situation that 0.25ng/ml TNF α exists;, record half and effectively protect dosage ED as positive control with Etanercept
50Be about 1-10ng/ml.
2. the active mensuration of adiponectin:
A) reference literature (Yokota T, Oritani K, Takahashi I, et al. (2000) Adiponectin, a newmember of the family of soluble defense collagens, negatively regulates the growth ofmyelomonocytic progenitors and the functions of macrophages.Blood 96:1723-1732.), record half and effectively suppress dosage ED
50Be about 10 μ g/ml.
B) measure sTNFR II-gAD (5 μ g/g body weight, abdominal injection) hypoglycemic effect to the back type i diabetes C57BL/6 mouse of taking food (sees the following form, n=5, with Walaphage as positive control), the result shows that sTNFR II-gAD has remarkable hypoglycemic effect in injection in back 4 hours.
The injection back time (hour) | 2 | 4 | 6 | 8 |
Physiological saline (%) | 9.10±3.58 | 16.50±5.47 | 33.25±4.66 | 45.25±3.09 |
Walaphage (%) | 45.00±7.36 * | 67.50±2.02 ** | 70.50±3.79 * | 67.00±9.14 |
sTNFRII- gAD(%) | 44.30±10.90 * | 42.50±8.17 * | 51.50±7.41 | 55.00±7.23 |
Annotate: * is P<0.05, and * * is P<0.01.
Soluble tumor necrosis factor receptor II-" adiponectin " bulb fusion rotein
<110〉Gao Jimin
<120〉soluble tumor necrosis factor receptor II-" adiponectin " bulb fusion rotein
<160>1
<210>1
<211>1023
<212>DNA
<213〉artificial sequence
<220>
<221>sTNFRII-gAD-6His
<222>(1)…(555)
<223〉this dna fragmentation is the cDNA of coding human soluble tumor necrosis factor receptor II (sTNFRII), and wherein 5 ' end contains ATG, as the initiation codon of intestinal bacteria translation sTNFRII-gAD-6His fusion rotein.
<220>
<221>sTNFRII-gAD-6His
<222>(556)…(564)
<223〉be the restriction enzyme site of EcoRI and NdeI mend flat after, flush end is connected to form.
<220>
<221>sTNFRII-gAD-6His
<222>(565…1023)
<223〉dna fragmentation (459bp) coding " adiponectin " bulb (gAD), its 3 ' end contains XhoI site of coding, the sequence (997-1023bp) of 6 Histidines and stop code.
<400>1
1 ATGTTGCCCGCCCAGGTGGCATTTACACCCTACGCCCCGGAGCCCGGGAGCACATGCCGG 60
MetLeuProAlaGlnValAlaPheThrProTyrAlaProGluProGlySerThrCysArg
61 CTCAGAGAATACTATGACCAGACAGCTCAGATGTGCTGCAGCAAATGCTCGCCGGGCCAA 120
LeuArgGluTyrTyrAspGlnThrAlaGlnMetCysCysSerLysCysSerProGlyGln
121 CATGCAAAAGTCTTCTGTACCAAGACCTCGGACACCGTGTGTGACTCCTGTGAGGACAGC 180
HisAlaLysValPheCysThrLysThrSerAspThrValCysAspSerCysGluAspSer
181 ACATACACCCAGCTCTGGAACTGGGTTCCCGAGTGCTTGAGCTGTGGCTCCCGCTGTAGC 240
ThrTyrThrGlnLeuTrpAsnTrpValProGluCysLeuSerCysGlySerArgCysSer
241 TCTGACCAGGTGGAAACTCAAGCCTGCACTCGGGAACAGAACCGCATCTGCACCTGCAGG 300
SerAspGlnValGluThrGlnAlaCysThrArgGluGlnAsnArgIleCysThrCysArg
301 CCCGGCTGGTACTGCGCGCTGAGCAAGCAGGAGGGGTGCCGGCTGTGCGCGCCGCTGCGC 360
ProGlyTrpTyrCysAlaLeuSerLysGlnGluGlyCysArgLeuCysAlaProLeuArg
361 AAGTGCCGCCCGGGCTTCGGCGTGGCCAGACCAGGAACTGAAACATCAGACGTGGTGTGC 420
LysCysArgProGlyPheGlyValAlaArgProGlyThrGluThrSerAspValValCys
421 AAGCCCTGTGCCCCGGGGACGTTCTCCAACACGACTTCATCCACGGATATTTGCAGGCCC 480
LysProCysAlaProGlyThrPheSerAsnThrThrSerSerThrAspIleCysArgPro
481 CACCAGATCTGTAACGTGGTGGCCATCCCTGGGAATGCAAGCATGGATGCAGTCTGCACG 540
HisGlnIleCysAsnValValAlaIleProGlyAsnAlaSerMetAspAlaValCysThr
541 TCCACGTCCCCCACCGAATTTATGAAAGGAGAACCTGGAGAAGGTGCCTATGTATACCGC 600
SerThrSerProThrGlTPheMetLysGlyGlTProGlyGlTGlyAlaTyrValTyrArg
601 TCAGCATTCAGTGTGGGATTGGAGACTTACGTTACTATCCCCAACATGCCCATTCGCTTT 660
SerAlaPheSerValGlyLeTGlTThrTyrValThrIleProAsnMetProIleArgPhe
661 ACCAAGATCTTCTACAATCAGCAAAACCACTATGATGGCTCCACTGGTAAATTCCACTGC 720
ThrLysIlePheTyrAsnGlnGlnAsnHisTyrAspGlySerThrGlyLysPheHisCys
721 AACATTCCTGGGCTGTACTACTTTGCCTACCACATCACAGTCTATATGAAGGATGTGAAG 780
AsnIleProGlyLeTTyrTyrPheAlaTyrHisIleThrValTyrMetLysAspValLys
781 GTCAGCCTCTTCAAGAAGGACAAGGCTATGCTCTTCACCTATGATCAGTACCAGGAAAAT 840
ValSerLeTPheLysLysAspLysAlaMetLeTPheThrTyrAspGlnTyrGlnGlTAsn
841 AATGTGGACCAGGCCTCCGGCTCTGTGCTCCTGCATCTGGAGGTGGGCGACCAAGTCTGG 900
AsnValAspGlnAlaSerGlySerValLeTLeTHisLeTGlTValGlyAspGlnValTrp
901 CTCCAGGTGTATGGGGAAGGAGAGCGTAATGGACTCTATGCTGATAATGACAATGACTCC 960
LeTGlnValTyrGlyGlTGlyGlTArgAsnGlyLeTTyrAlaAspAsnAspAsnAspSer
961 ACCTTCACAGGCTTTCTTCTCTACCATGACACCAACCTCGAGCACCACCACCACCACCAC 1020
ThrPheThrGlyPheLeTLeTTyrHisAspThrAsnLeuGluHisHisHisHisHisHis
1021 TGA 1023
*
Claims (3)
1. fusion rotein, it is characterized in that fusion rotein that the gene of soluble tumor necrosis factor receptor II (sTNFR II) and " adiponectin " bulb (gAD) gene are produced through gene recombination, expression, i.e. solubility Tumor Necrosis Factor Receptors II-" adiponectin " bulb fusion rotein (sTNFR II-gAD fusion rotein).
2. fusion rotein according to claim 1 is characterized in that fusion gene cloning being built into sTNFR II-gAD-pET24 expression plasmid in the expression vector pET24 that has the T7 promotor, and transformed into escherichia coli BL21 (DE3) realizes the back efficiently expressing; The engineering bacteria that transforms the back acquisition is intestinal bacteria sTNFR II-gAD/pET24.
3. fusion rotein according to claim 1 and 2 is characterized in that the C that 6His is introduced fusion rotein holds.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102690353A (en) * | 2011-03-25 | 2012-09-26 | 温州医学院 | Method for rapid screening of modified tumor necrosis factor receptor II-adiponectin globular-site fusion protein and application thereof |
CN101906156B (en) * | 2010-02-09 | 2012-10-10 | 中国药科大学 | Structures and application of bifunctional protein and derivatives of bifunctional protein |
WO2022188883A1 (en) * | 2021-03-12 | 2022-09-15 | 上海赛金生物医药有限公司 | Fusion protein of tnfr2 and april baff receptor |
Family Cites Families (4)
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CN1204252C (en) * | 2001-10-31 | 2005-06-01 | 上海中信国健药业有限公司 | Recombinant gene of soluble part in tumor necrosis factor acceptor and its fusion gene and product |
CN1246337C (en) * | 2002-11-26 | 2006-03-22 | 广州绿阳生物工程有限公司 | Novel TNFR-Fc fusion protein |
EP1636264A2 (en) * | 2003-06-24 | 2006-03-22 | MERCK PATENT GmbH | Tumour necrosis factor receptor molecules with reduced immunogenicity |
CN1565631A (en) * | 2003-06-27 | 2005-01-19 | 上海中信国健药业有限公司 | Usage of fused protein of TNF receptor and globin in acute lung injury treating medicine |
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2005
- 2005-10-20 CN CNB2005101004689A patent/CN1309740C/en active Active
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101906156B (en) * | 2010-02-09 | 2012-10-10 | 中国药科大学 | Structures and application of bifunctional protein and derivatives of bifunctional protein |
CN102690353A (en) * | 2011-03-25 | 2012-09-26 | 温州医学院 | Method for rapid screening of modified tumor necrosis factor receptor II-adiponectin globular-site fusion protein and application thereof |
CN102690353B (en) * | 2011-03-25 | 2014-05-14 | 温州医学院 | Method for rapid screening of modified tumor necrosis factor receptor II-adiponectin globular-site fusion protein and application thereof |
WO2022188883A1 (en) * | 2021-03-12 | 2022-09-15 | 上海赛金生物医药有限公司 | Fusion protein of tnfr2 and april baff receptor |
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Effective date of registration: 20090206 Address after: Dasan Zhejiang city of Wenzhou Province Higher Education Park Patentee after: Wenzhou Medical College Address before: Study on 13 biological drug gang Lu Guangzhou province Guangdong City Chigang pomegranate Institute Patentee before: Gao Jimin |
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Owner name: WENZHOU MEDICAL COLLEGE Free format text: FORMER OWNER: GAO JIMIN Effective date: 20090206 |