CN102206267A - Molecular structure and application of tumor necrosis factor receptor 1 proligand assembly domain glycocluster - Google Patents
Molecular structure and application of tumor necrosis factor receptor 1 proligand assembly domain glycocluster Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and specially relates to a molecular structure, a preparation method and an application of a tumor necrosis factor receptor 1 proligand assembly domain glycocluster. A proligand assembly domain (PLAD) of a tumor necrosis factor receptor 1 and molecules of polyethylene glycol (PEG) and the like are connected through covalent bonds to form a tumor necrosis factor receptor 1 proligand assembly domain protein glycocluster molecule, wherein the PLAD part can be prepared through a technology of protein recombinant and expression. The invention aims at obtaining a PLAD molecule with a long serum half-life period and the PLAD molecule is utilized for treating human inflammatory diseases.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of Tumor Necrosis Factor Receptors 1 preceding part assembling territory protein conjugate.
Background technology
The extracellular region of Tumor Necrosis Factor Receptors (TNFR) superfamily mainly be responsible for the identification of aglucon with combine, their extracellular region contains 1 to 6 structural domain (CRD) that is rich in halfcystine usually.These acceptors are brought into play function of receptors usually with the tripolymer binding partner.Above-mentioned functions is mediated by containing 1 preceding part assembling territory of being rich in halfcystine at least.The TNFR superfamily member comprises that TRAIL acceptor 1, CD40, TNFR1 and TNFR2 possess this association function.Other TNFR superfamily members such as Fas, LT β R, HVEM, RANK, CD40, CD30, CD27, OX40 and DR4 all contain the territory of part assembling before this.
Discover, lacking under the situation of aglucon that polymeric formation is necessary and sufficient to PLAD to acceptor.Though PLAD do not participate in directly to the identification of TNF α and TNF β with combine, deletion PLAD or PLAD undergo mutation, the affinity of part and acceptor can be lost.Therefore, PLAD for the polymeric formation of TNFR and aglucon in conjunction with relevant, the conduction of TNF coherent signal by way of in play crucial effects.The TNFR superfamily member is by preformed polymer rather than the crosslinked signal that transmits by the single receptor subunit of part inductive, so PLAD can be sealed to block these polymeric formation and therefore to block function of receptors by target.The discovery of PLAD with and the illustrating of aglucon independent form acceptor composition function of mediation be that the treatment of relative disease provides new approaches and novel targets.
Listing at present with TNF α or TNFR be the bio-pharmaceutical of the treatment rheumatoid arthritis of target spot have Infliximab (
Ying Fuli former times), Adalimumab (
Adalimumab) and Etanercept (
Etanercept) etc.Sharp former times of its Sino-British husband and adalimumab are the monoclonal antibodies of TNF α, and etanercept is soluble TNF R and the segmental fusion rotein of Fc.TNF α monoclonal antibody and TNFR fusion rotein can be in conjunction with excessive soluble TNF α, thereby suppress the signal transduction pathway that TNF α causes.But since this be that the methods of treatment of target spot can not be distinguished multiple TNFR path with TNF α, such as the combination that can not distinguish TNF α and TNFR1 and TNFR2, so when the disease that inhibition TNFR1 causes, also can suppress the physiological action of TNFR2 mediation, thereby may cause multiple side effect, as the increase of lupoid acne syndromes (lupus-like disease), mycobacterial infections and lymphoma incidence.
If with PLAD is target spot, use the PLAD district of sealing TNFR such as PLAD or its functional mutants, can suppress the polymerization of certain TNFR specifically, thereby stop the formation of TNFR-TNF mixture, reach the purpose of termination signal conduction.Because every kind of acceptor only and the PLAD district combination of self, so this combination has specificity.When disturbing the function of TNFR1, can avoid influence, thereby avoid the generation of side effect the TNFR2 physiological function such as the PLAD that uses TNFR1.
Polyoxyethylene glycol (PEG) is the polymerization macromole of a class non-immunogenicity, nontoxic, good water solubility, and polyethyleneglycol modified technology is the method that is widely used in improving protein biological effectiveness and physico-chemical property now.Modified the protein molecule of polyoxyethylene glycol since can reduce its by the palliating degradation degree of proteolytic enzyme, reduce glomerular filtration rate(GFR, increase proteic water-soluble and reduce the immunogenicity of protein molecule, therefore can be endowed better biology and physicochemical property, enlarge the range of application of protein and polypeptide.Wherein since the flexibility of PEG molecule long-chain with and bigger molecular weight, the transformation period can effectively be prolonged in the protein body in vivo after PEG modifies, and delays its intravital clean-up time, and can effectively improve proteinic bio distribution.At present, the protein drug that the multiple PEG of FDA approved modifies enters the biological medicine Application Areas, the adenosine deaminase of modifying as PEG (PEG-ADA), asparaginase (PEG-L-asparaginase), Interferon Alpha-2b (peginterferon alfa-2b), Intederon Alpha-2a (peginterferon alfa-2a), recombinant methionyl human G-CSF (pegfilgrastim) etc.
PEG modifies by chemical reaction takes place between protein molecule and the activated PEG modifier and finishes.The multiple group that can be connected with peg moiety is arranged on the amino acid chain, as-NH
2,-NH-,-COOH ,-OH ,-SH and disulfide linkage etc.Wherein amido modified is that the PEG that is most widely used at present modifies means, comprises the ε-NH of Methionin
2α-NH with the protein N end
2Be the maximum nucleophilic group of quantity in protein and the polypeptide, and usually be exposed to the surface of molecule, therefore be easy to modify reagent react with PEG.At present, being used for amido modified polyethyleneglycol modified dose mainly contains following several: PEG-succinimide succinate (PEG-SS), PEG-succinimdyl carbonate (PEG-SC), PEG-trifluoroethyl sulphonate (PEG tresylate) and PEG-aldehyde (PEG aldehyde) etc., the structure formation of its PEG poly chain also comprises straight chain and branch-like.But, if the amido modified ε-NH that comprises Methionin of protein or polypeptide
2α-NH with the N-end
2, the modification regular meeting of esters of gallic acid activating group obtains the reaction product of multiple different modifying site and degree of modification.The reduction alkylation reaction of protein and PEG-aldehyde then can overcome this deficiency, because the terminal α-NH of protein N
2The pKa value be lower than the ε-NH of Methionin
2The pKa value, therefore in weak acid environment, the easier α-NH with terminal amino acid residue of PEG-aldehyde
2The reduction alkylation reaction takes place, thereby realizes the pointed decoration of N-terminal amino group.In addition, introduce alpha-non-natural amino acid by genetic engineering technique, as to acetylphenylalanine or to the phenylazide L-Ala, at the specific groups on the alpha-non-natural amino acid, adopting the polyethyleneglycol derivative with corresponding activating group that it is carried out coupling also is the present comparatively strategy of pointed decoration commonly used again.
Summary of the invention
Part assembles the territory protein conjugate before the objective of the invention is to obtain a kind of Tumor Necrosis Factor Receptors 1.For achieving the above object, the present invention has adopted following technological line and step:
One, technological line
1. part assembles the territory protein conjugate before the chemosynthesis Tumor Necrosis Factor Receptors 1; Perhaps according to the proteic gene in part assembling territory before the synthetic Tumor Necrosis Factor Receptors 1 of corresponding host's codon-bias.
2. expression vector is gone in gene recombination, carries out abduction delivering after being cloned into the host bacterium.
3. separation and purification obtains target protein.
4. the coupling of part assembling territory albumen and peg molecule before the Tumor Necrosis Factor Receptors 1.
5. part assembled the territory protein conjugate before separation and purification obtained Tumor Necrosis Factor Receptors 1 from the linked reaction mixture.
6. the evaluation and the application of part assembling territory protein conjugate before the Tumor Necrosis Factor Receptors 1.
Part assembling territory albumen before the Tumor Necrosis Factor Receptors 1 of the present invention, be PLAD structural domain and the functional variant thereof that comes from Tumor Necrosis Factor Receptors 1 extracellular region, part assembling territory albumen has identity before the aminoacid sequence of mutant about at least 90% and the Tumor Necrosis Factor Receptors in natural human source 1.
The part assembling proteic acquisition in territory can be expressed by chemosynthesis or protein recombinant technology and obtain before the Tumor Necrosis Factor Receptors 1 involved in the present invention.Protein recombinant expression system of the present invention can be procaryotic cell expression system or eukaryotic cell expression system.Preferred procaryotic cell expression system by cutting except that fusion tag with soluble protein label amalgamation and expression and proteolytic enzyme such as enteropeptidase enzyme, obtains part assembling territory albumen before the Tumor Necrosis Factor Receptors 1 in a large number.
Contain alpha-non-natural amino acid as can realizing by genetic code extended technology fixedpoint introduction of non-natural amino acid to the phenylazide L-Ala or to the part assembling proteic acquisition in territory before the Tumor Necrosis Factor Receptors 1 of acetylphenylalanine.
Above-mentioned polyoxyethylene glycol can be linear chain structure or branched structure.Molecular weight ranges is 2000-40000Da.
The preparation method of part assembling territory protein conjugate is before the Tumor Necrosis Factor Receptors 1 of the present invention: use damping fluid, regulate PLAD protein solution pH value to 3.0 to 9.0, add polymkeric substance such as mPEG-aldehyde and catalyzer (as sodium cyanoborohydride) then in proportion with activating group, leave standstill or the stirring reaction regular hour, reaction is stopped reaction mixture dilution or adjusting pH value, adopt ion-exchange or gel filtration chromatography purifying PEG-PLAD molecule.
Above-mentioned damping fluid can be acetic acid-sodium-acetate buffer, boric acid-borate buffer solution, phosphoric acid buffer, carbonic acid-sodium carbonate buffer, tris-HCI buffer, phosphate buffered saline buffer or sodium citrate buffer solution etc.The mol ratio of protein and polyoxyethylene glycol is between 1: 1 to 1: 10, and temperature of reaction is 0-37 degree centigrade, and the reaction times is more than 15 minutes.
Part assembles the territory protein conjugate before the objective of the invention is to obtain a kind of Tumor Necrosis Factor Receptors 1, compare with PLAD prototype molecule, conjugate has stability and long interior transformation period of body preferably, can overcome PLAD prototype molecule easily by the deficiency of enzymolysis and removing, simultaneously the same with PLAD prototype molecule, the activity that possesses TNFR related ligands such as anti-TNF alpha short of money, because TNF α is the important factor that causes human panimmunity inflammation, so this conjugate is expected to be applied to treat as human inflammatory diseasess such as rheumatoid arthritiss.
Description of drawings
Fig. 1 shows that Trcine-SDS-PAGE analyzes the TNFR1-PLAD purity of protein that obtains behind the purifying
Sequence number explanation among the figure: 1. protein molecular weight standard; 2. the TNFR1-PLAD albumen that obtains behind the purifying
Fig. 2 obtains the TNFR1-PLAD purity of protein after showing HPLC analysis purifying
Fig. 3 shows that SDS-PAGE analyzes mPEG-ALD20000 and modifies the product that obtains behind the PLAD
Sequence number explanation among the figure: 1.mPEG-ALD20000 modifies the reaction solution of PLAD; 2. protein molecular weight standard
Embodiment
Below in conjunction with embodiment, be described in further detail the present invention.Method among specification sheets and the embodiment, as NOS all routinely experiment condition operate, or operate by the explanation that supplier provides.
MPEG-aldehyde and PLAD (SEQ ID NO:1, N-terminal α amino coupled Xaa=Leu)
Use acetic acid-sodium-acetate buffer of 20mM that PLAD is configured to 10mg/ml, the protein soln of pH5.2, add the methoxy poly (ethylene glycol) propionic aldehyde (mPEG-ALD20000) of the molecular weight of acetic acid-sodium-acetate buffer configuration of using 20mM inward as 20000Da, make that the mol ratio of protein and PEG is 1: 1, adding final concentration is the sodium cyanoborohydride reduction catalysts reaction of 20mM.In 4 degrees centigrade of standing and reacting 16 hours.After reaction finished, dilute reaction solution was also regulated the pH value to neutral stopped reaction.Reaction is undertaken by following reaction formula:
MPEG-maleimide and PLAD (SEQ ID NO:2, Xaa=Cys) Mo Duan Cys coupling
Use the Tris-HCl damping fluid of 20mM that PLAD is configured to 6mg/ml, the protein soln of pH8.0, add the methoxy poly (ethylene glycol) maleimide (mPEG-MAL2000) of the molecular weight of the Tris-HCl damping fluid configuration of using 20mM inward, make that the mol ratio of protein and PEG is 1: 1 as 2000Da.In 4 degrees centigrade of standing and reacting more than 6 hours.
Reaction is undertaken by following reaction formula:
Embodiment 3
MPEG-vinyl sulphone and PLAD (SEQ ID NO:3, Xaa=Cys) Mo Duan Cys coupling
Use the Tris-HCl damping fluid of 20mM that PLAD is configured to 6mg/ml, the protein soln of pH8.0, add the methoxy poly (ethylene glycol) vinyl sulphone (mPEG-VS5000) of the molecular weight of the Tris-HCl damping fluid configuration of using 20mM inward, make that the mol ratio of protein and PEG is 1: 2 as 5000Da.In 4 degrees centigrade of standing and reacting more than 6 hours.Reaction is undertaken by following reaction formula:
MPEG-iodo-acid amide and PLAD (SEQ ID NO:4, Xaa=Cys) Mo Duan Cys coupling
Use the Tris-HCl damping fluid of 20mM that PLAD is configured to 5mg/ml, the protein soln of pH8.0, add the methoxy poly (ethylene glycol) iodo-acid amide (mPEG-IA10000) of the molecular weight of the Tris-HCl damping fluid configuration of using 20mM inward, make that the mol ratio of protein and PEG is 1: 10 as 10000Da.In 4 degrees centigrade, the lucifuge standing and reacting is more than 10 hours.Reaction is undertaken by following reaction formula:
Embodiment 5
The adjacent pyridine disulfide of mPEG-and PLAD (SEQ ID NO:1, Xaa=Cys) Mo Duan Cys coupling
Use acetic acid-sodium-acetate buffer of 20mM that PLAD is configured to 10mg/ml, the protein soln of Ph5.8, the molecular weight that adds acetic acid-sodium-acetate buffer configuration of using 20mM inward makes that as the adjacent pyridine disulfide (mPEG-OPSS30000) of the methoxy poly (ethylene glycol) of 30000Da the mol ratio of protein and PEG is 1: 5.In 4 degrees centigrade of standing and reacting more than 3 hours.The disulfide linkage that reaction forms can be reduced the agent reduction, so avoid contacting reductive agent in the reaction process.Reaction is undertaken by following reaction formula:
Embodiment 6
MPEG-hydrazides and PLAD (SEQ ID NO:2, Xaa=pAcF) Mo Duan pAcF coupling
Use the phosphate buffered saline buffer of 20mM that PLAD is configured to 3mg/mL, the protein soln of pH7.0, add the methoxy poly (ethylene glycol) hydrazides (mPEG-hydrazide20000) of the molecular weight of the phosphate buffered saline buffer configuration of using 20mM inward, make that the mol ratio of protein and PEG is 1: 3 as 20000Da.In 4 degrees centigrade of standing and reacting 24 hours.Reaction is undertaken by following reaction formula:
Embodiment 7
MPEG-alkynes and PLAD (SEQ ID NO:3, Xaa=pAzF) Mo Duan pAzF coupling
Use boric acid-borate buffer solution of 20mM that PLAD is configured to 1mg/mL, the protein soln of pH8.4, add the methoxy poly (ethylene glycol) alkynes (mPEG alkyne20000) of the molecular weight of boric acid-borate buffer solution configuration of using 20mM inward, make that the mol ratio of protein and PEG is 1: 2 as 20000Da.The TCEP that adds CuSO4 that final concentration is 1mM and final concentration 2mM simultaneously was as catalyzer, in 4 degrees centigrade of standing and reacting 48 hours.Reaction is undertaken by following reaction formula:
Embodiment 8
The N-terminal α amino coupled of the mPEG-aldehyde of side chain and PLAD (SEQ ID NO:4, Xaa=disappearance)
Use acetic acid-sodium-acetate buffer of 20mM that PLAD is configured to 10mg/ml, the protein soln of pH5.6, add the branched chair polymacrogol aldehyde (Aldehyde PEGs 40000) of the molecular weight of acetic acid-sodium-acetate buffer configuration of using 20mM inward as 40000Da, make that the mol ratio of protein and PEG is 1: 1, adding final concentration is the sodium cyanoborohydride reduction catalysts reaction of 20mM.In 4 degrees centigrade of standing and reacting more than 6 hours.After reaction finished, dilute reaction solution was also regulated the pH value to neutral stopped reaction.Reaction is undertaken by following reaction formula:
Embodiment 9
PEG modifies the separation and purification of the mono-modified product in back
Use the balance liquid pre-equilibration SP Sepharose Fast Flow cationic exchange coloum of the pH6.8 of 10 times of column volumes, regulate PEG-aldehyde and PLAD reaction solution to pH value 6.8, after sample finishes on the speed of 2ml/min, wash pillar to the baseline balance with the balance liquid stream of the pH6.8 that does not contain NaCl with the flow velocity of 2ml/min.Use contains the balance liquid of 1MNaCl pH6.8 as B liquid, and the balance liquid that does not contain NaCl is as A liquid, and gradient is mixed wash-out, and B is increased to 100%, flow velocity 1ml/min from 0% in 50 minutes.Detect OD280 place light absorption value with AKTA protein preparation system, collect elution peak and be used for the subsequent analysis evaluation.
The reaction solution separation and purification process of embodiment 1-8 is all similar to aforesaid method, and only the elution time of component is variant, collects elution peak according to the AKTA detection case.
The mono-modified product of PEG that the reaction solution separation and purification of use embodiment 1-8 obtains is called after PEG-PLAD01, PEG-PLAD02, PEG-PLAD03, PEG-PLAD04, PEG-PLAD05, PEG-PLAD06, PEG-PLAD07 and PEG-PLAD08 respectively.
Biological activity is analyzed
L929 cultivates in the DMEM high glucose medium that contains 10% calf serum, adds the 1-2ml trysinization, observes cell rounding, shrinkage.Topple over and pancreatin, adding substratum piping and druming re-suspended cell.Use cell counting count board to calculate cell concn, use substratum that cell suspension is diluted to suitable concentration.100 μ l cell suspensions add in the 96 porocyte plates, and 37 ℃, the 5%CO2 overnight incubation makes cell attachment stable to growth conditions.Add the product to be tested of 25 μ l dactinomycins (final concentration is 2 μ g/ml) and 50 μ l gradient dilutions, 37 ℃, 5%CO2 is hatched 2h.Adding 25 μ l final concentrations is the TNF α of 1ng/ml, and 37 ℃ are continued to cultivate 16-18h.Add 20 μ lMTT solution, 37 ℃, 5%CO2 is hatched 4h.Careful sucking-off cell culture supernatant adds 150 μ lDMSO, and rocker machine 200rpm jolts the 5min mixing, uses microplate reader to measure light absorption value in 490nm or 570nm place.Calculating cell growth rate is G=(A
S-A
0)/A
0, A wherein
SBe sample well concentration, A
0Concentration for the blank hole.Adopt the SigmaPlot software analysis to calculate the ED50 value.The result is as shown in the table:
Embodiment 11
Lipidated protein is analyzed
(1)Tricine-SDS-PAGE
The Tricine-SDS-PAGE electrophoresis is fit to the little peptide analysis of molecular weight less than 10kDa.Main method is with reference to Hermann
Document.Dispose 16% separation gel, 10% gap glue and 4% concentrated glue respectively.Constant voltage 30V behind the last sample, after sample entered gap glue, voltage rose to 80V, and after sample entered separation gel, constant voltage 100V finished to electrophoresis.Gel fixedly behind the 30min, adopts Coomassie brilliant blue G250 dyeing through stationary liquid, and 10% Glacial acetic acid takes off to background transparent as destainer.
(2)SDS-PAGE
SDS-PAGE is used to analyze the PLAD after PEG modifies.Main method is with reference to " molecular cloning ", and preparation 5% concentrates glue, 15% separation gel, and constant voltage 80V finishes to electrophoresis behind the last sample.After the staining fluid dyeing of gel through containing Coomassie brilliant blue G250, decolour to background transparent.
(3)HPLC
HPLC analyzes and adopts SHIMADZU LC-2010C HT liquid chromatographic system, chromatographic column: SHIMADZU VP-ODS150L * 4.6.Concrete grammar is as follows: mobile phase A: water, and Mobile phase B: acetonitrile (containing 0.1% trifluoroacetic acid), flow velocity: 1ml/min, column temperature: 30 ℃, gradient elution, 5%B is to 95%B in 35 minutes.Detect 280nm place light absorption value.
Claims (5)
1. Tumor Necrosis Factor Receptors 1 a preceding part assembling territory protein conjugate is characterized in that comprising A and the B two portions that connect by covalent linkage.
Wherein A is the preceding part assembling territory (PLAD) and the functional variant thereof of Tumor Necrosis Factor Receptors 1 extracellular region, comprises sequence 1 (SEQ ID NO:1), sequence 2 (SEQID NO:2), sequence 3 (SEQID NO:3) or sequence 4 (SEQ ID NO:4) aminoacid sequence shown in one of them;
Sequence 1:
Xaa?Val?Pro?His?Leu?Gly?Asp?Arg?Glu?Lys?Arg?Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys?TyrIle?His?Pro?Gln?Asn?A sn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?AsnAsp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?As
Wherein: Xaa is: Leu, Cys, pAzF, pAcF;
Sequence 2:
Leu?Val?Pro?His?Leu?Gly?Asp?Arg?Glu?Lys?Arg?Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys?TyrIle?His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?AsnAsp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Xaa
Wherein: Xaa is: Arg, Cys, pAzF, pAcF;
Sequence 3:
Xaa?Leu?Val?Pro?His?Leu?Gly?Asp?Arg?Glu?Lys?Arg?Asp?Ser?Val?Cys?Pro?Gln?Gly?LysTyr?Ile?His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?TyrAsn?Asp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg
Wherein: Xaa is: Cys, pAzF, pAcF or disappearance;
Sequence 4:
Leu?Val?Pro?His?Leu?Gly?Asp?Arg?Glu?Lys?Arg?Asp?Ser?Val?Cys?Pro?Gln?Gly?Lys?TyrIle?His?Pro?Gln?Asn?Asn?Ser?Ile?Cys?Cys?Thr?Lys?Cys?His?Lys?Gly?Thr?Tyr?Leu?Tyr?AsnAsp?Cys?Pro?Gly?Pro?Gly?Gln?Asp?Thr?Asp?Cys?Arg?Xaa
Wherein: Xaa is: Cys, pAzF, pAcF or disappearance;
Above-mentioned pAzF representative is to the phenylazide L-Ala, and pAcF represents acetylphenylalanine.
B is the polymkeric substance that can improve A stability.
2. conjugate according to claim 1 is characterized in that B is: polyoxyethylene glycol, dextran, polyethylene, polypropylene, polyvinyl alcohol, polysialic acids, palmitinic acid or lauric acid one of them.
3. conjugate according to claim 2 is characterized in that B is a polyoxyethylene glycol, and its molecular weight ranges is 2000 to 40000Da.
4. conjugate according to claim 2 is characterized in that forming the covalent linkage formation by one of them terminal activating group of the adjacent pyridine disulfide of mPEG-aldehyde, mPEG-maleimide, mPEG-, mPEG-vinyl sulphone, mPEG-iodo-acid amide, mPEG-hydrazides or mPEG-alkynes with the A reaction.
5. conjugate according to claim 1 is characterized in that being used for the treatment of human inflammatory disorders such as rheumatoid arthritis.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107073133A (en) * | 2014-10-27 | 2017-08-18 | 吉利斯拉有限公司 | Material and method on the joint for being used in protein drug conjugate |
| CN110551195A (en) * | 2019-08-01 | 2019-12-10 | 天津科技大学 | Alpha-synuclein aggregation-induced emission system and construction and application thereof |
-
2011
- 2011-04-18 CN CN2011100958500A patent/CN102206267A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107073133A (en) * | 2014-10-27 | 2017-08-18 | 吉利斯拉有限公司 | Material and method on the joint for being used in protein drug conjugate |
| US11285220B2 (en) | 2014-10-27 | 2022-03-29 | Iksuda Therapeutics Limited | Materials and methods relating to linkers for use in protein drug conjugates |
| CN110551195A (en) * | 2019-08-01 | 2019-12-10 | 天津科技大学 | Alpha-synuclein aggregation-induced emission system and construction and application thereof |
| CN110551195B (en) * | 2019-08-01 | 2022-08-09 | 天津科技大学 | Alpha-synuclein aggregation-induced emission system and construction and application thereof |
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Application publication date: 20111005 |