CN101747440B - TNFR-Fc fusion protein and usage thereof - Google Patents
TNFR-Fc fusion protein and usage thereof Download PDFInfo
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Abstract
The invention relates to a TNFR-Fc fusion protein and usage thereof. By adopting molecular biological technology, cell biological technology and immunological technology, the invention builds a fusion protein of soluble segment of human TNFa receptor and Fc segment of human IgG2. The fusion protein can be used for the prevention or treatment of diseases related to TNFa abnormal activation. The fusion protein has the advantages that the TNFa combination effect is extremely excellent, the stability is good, the half life is long and the side effect is low.
Description
Technical field
The invention belongs to biology field, relate to a kind of new restructuring TNFR-Fc fusion rotein and uses thereof.
Background technology
Human tumor necrosis factor (hTNF-α) is mainly the cytokine being produced by a kind of Monocytes/Macrophages of activation, this is a kind of cytokine with various biological effect, TNF-α energy inducing tumor cell necrosis or apoptosis are found in early stage research, but found afterwards that TNF-α was the major cytokine of inducing inflammatory reaction, also be the important factor that causes heating and septic shock, in heart failure, graft-rejection and autoimmune disease, play an important role.Appropriate TNF-α energy activating immune system, strengthening immunity, resist in the system of defense that microorganism is invaded and inhibition tumour produces and play keying action host.But during excessive TNF-alpha expression, can produce multiple pathology damage together with other inflammatory factor.Therefore, TNF-α plays a part double-edged sword in vivo.
People have confirmed that the level of TNF-α is obviously high than normal people in the research of various diseases, and find its possible pathogenesis (Feldman M, et al., Ann Rev Immunol 1996; 14:397).Because TNF-α has participated in generation and the evolution of various diseases, in some disease, play a crucial role, in some disease with other factors acting in conjunction; Therefore on different levels, block the effect of TNF-α, likely the disease relevant with TNF-α produced to therapeutic action.The at present treatment disease take TNF-α as target, what have gets permission clinical use, has obtained expected effect, and what have carries out clinical experiment.
There are two kinds of different TNFR in cell surface, molecular weight is respectively the TNFRI (CD120 α) of 55KD and the TNFRII (CD120 β) of 75KD.The avidity of humanTNF-α and TNFR is higher, is respectively 1.23 ± 0.23nM and 0.36 ± 0.13nM with the affinity costant of TNFRI and TNFRII.The film outskirt of amphitypy TNFR is all tear-away, still retains the activity in conjunction with TNF-α.The film outskirt of acceptor is connected with the Fc section of antibody, and the fusion rotein of formation both can be combined with TNF-α, had increased again stability, and can form dimer by Fc section, than the monomeric acceptor of natural appearance, TNF-α was had to larger avidity.
In prior art, American I mmunex company has developed commercial TNFRII-IgG1/Fc fusion rotein, is called Etanercept, and commodity are called Enbrel,, by FDA approval listing, be used for the treatment of bone and the joint injury of rheumatoid arthritis, ankylosing spondylitis and inhibition psoriasis patients.But, comprise Enbrel at the TNF of interior multiple listing molecule agonist drug, due to these medicines there is ADCC and CDC effect in inductor may (Tracey D, et al., Pharmacol Ther.2008; 117 (2): 244-79), all there is certain side effect in these medicines therefore.
But the preparation of the combination TNF α finding at present also exists binding ability lower, biological activity is not high, body interior effective acting time of short defect, and therefore this area is necessary the medicine of further Improvement, effectively to improve curative effect of medication.
Summary of the invention
The object of the present invention is to provide a kind of fusion rotein, the aminoterminal of described fusion rotein is the solvable fragment of human tumor necrosis factor II receptor (TNFR II); Carboxyl terminal is human IgG2's Fc fragment.
Another object of the present invention is to purposes and the composition of the fusion rotein that provides described.
In a first aspect of the present invention, a kind of fusion rotein is provided, the aminoterminal of described fusion rotein is the solvable fragment of human tumor necrosis factor II receptor (TNFR II); Carboxyl terminal is the Fc fragment of human normal immunoglobulin 2 (IgG2).
In another preference, the described fusion rotein transformation period was in vivo higher than 48 hours.
In another preference, described human tumor necrosis factor II receptor has the aminoacid sequence shown in SEQ ID NO:1.Preferably, the aminoacid sequence of the described solvable fragment of people II type TNF α acceptor is as shown in SEQ ID NO:1.
In another preference, described human IgG2's Fc fragment has the aminoacid sequence shown in SEQ ID NO:2.Preferably, the aminoacid sequence of described human IgG2's Fc fragment is as shown in SEQ ID NO:2.
In a second aspect of the present invention, provide a kind of nucleic acid molecule, the fusion rotein described in described nucleic acid molecule encoding.
In another preference, described nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:3.
In a third aspect of the present invention, a kind of carrier is provided, it contains described nucleic acid molecule.
In another preference, in described carrier, be also connected with operably the encoding gene of glutamine synthetase (GS).
In another preference, the aminoacid sequence of described glutamine synthetase is as shown in SEQ ID NO:4.
In another preference, the coding gene sequence of described glutamine synthetase is (or sequence information is as Genebank X03495) as shown in SEQ ID NO:5.
In another preference, described carrier is pIRES two-cistron expression vector.
In another preference, the coding gene sequence of described glutamine synthetase is positioned at the multiple clone site B of this expression vector; Above-mentioned nucleic acid molecule is positioned at the multiple clone site A of this expression vector.
In a fourth aspect of the present invention, a kind of genetically engineered cell is provided,
Described cell contains described carrier;
Or the nucleic acid molecule described in being integrated with in described cellular genome.
In a fifth aspect of the present invention, a kind of method that produces described fusion rotein is provided, described method comprises: being applicable to expressing under the condition of described fusion rotein, cultivate described host cell, express and isolate described fusion rotein.
In a sixth aspect of the present invention, the purposes of the fusion rotein described in providing, for the preparation of the composition of specific binding tumor necrosis factor alpha.
In another preference, described composition is for prevention or treatment tumor necrosis factor alpha abnormal activation relative disease.
In another preference, described tumor necrosis factor alpha abnormal activation relative disease comprises: rheumatoid arthritis, ankylosing spondylitis, psoriatic, septicemia, asthma, apoplexy, diabetes, Crohn ' s disease.
In a seventh aspect of the present invention, a kind of composition of specific binding tumor necrosis factor alpha is provided, described composition contains:
(i) the described fusion rotein of significant quantity; With
(ii) pharmaceutically acceptable carrier.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Fig. 1. the enzyme of recombinant plasmid is cut qualification result.Wherein, swimming lane 1 is DL250bp Marker; Swimming lane 2 is PCR checking recombinant plasmid; Swimming lane 3 is double digestion evaluation recombinant plasmid; Swimming lane 4 is DL500～15, the Marker of 000bp.
Fig. 2. the ELISA typical curve that utilizes TNFR-IgG2Fc standard substance to prepare.
Fig. 3. utilize separating medium rProtein A Sepharose 4 Fast Flow and Q Sepharose FF to carry out affinity chromatography and ion exchange chromatography, purifying expressing protein.
Fig. 4. the SDS-PAGE of the albumen after purifying detects.Wherein,
The non-reduced sample of swimming lane 3. (211p080723-3); The non-reduced sample of swimming lane 4. (211p080723-4);
The non-reduced sample of swimming lane 5. (Enbrel); Swimming lane 6. protein labelings;
Fig. 5. the western trace of the albumen after purifying detects.Wherein,
The non-reduced sample of swimming lane 3. (211p080723-3); The non-reduced sample of swimming lane 4. (211p080723-4);
The non-reduced sample of swimming lane 5. (Enbrel); Swimming lane 6. protein labelings;
Fig. 6 .TNF α in vitro toxicity is in conjunction with the baseline results of experiment.
Embodiment
The inventor is through deep research, be surprised to find that the solvable fragment of human tumor necrosis factor II receptor (TNFRII) and human IgG2's Fc fragment are merged mutually, the fusion rotein obtaining has the effect of extremely excellent combination TNF α, and good stability, long half time, side effect are low.
As used herein, unless otherwise indicated, TNFR, TNFR II, hTNFR II is used interchangeably, and all refers to human tumor necrosis factor II receptor.
As used herein, unless otherwise indicated, IgG2 Fc or hIgG/Fc refer to the Fc fragment of human normal immunoglobulin 2, and it is different from the Fc fragment (IgG1 Fc) of human normal immunoglobulin 1.
" containing " as used herein, described, " having " or " comprising " comprised " comprising ", " mainly by ... form ", " substantially by ... form " and " by ... form "; " mainly by ... form ", " substantially by ... form " and " by ... formation " belong to the subordinate concept of " containing ", " having " or " comprising ".
As used herein, unless otherwise indicated, described fusion rotein is a kind of albumen of separation, without contacting, is the purified product of recombinant host cell cultivation or the extract as a kind of purifying with other albumen, polypeptide or molecule.
The invention provides a kind of fusion rotein, comprise the solvable fragment of human soluble tumor necrosis factor II receptor, the Fc fragment (IgG2 Fc) of human normal immunoglobulin 2.This fusion rotein can be used in the composition of preparing specific binding tumor necrosis factor alpha.This fusion rotein is abbreviated as " TNFR-IgG2/Fc " or " hTNFR II-IgG2/Fc ".
In described fusion rotein, aminoterminal is the solvable fragment of TNFR II; Carboxyl terminal is IgG2 Fc fragment, and described IgG Fc fragment has hinge area, CH2He CH3 district.
TNFR-IgG2/Fc fusion rotein relative molecular weight of the present invention is 150 kilodaltons, and wherein amino-acid residue accounts for 100 kilodalton left and right, and all the other are sugar chain.Every peptide chain is containing 3 N glycosylation sites, and 2 polypeptide chains can form disulfide linkage by cysteine residues, the dimer such as becomes.Due to the member of TNFR II also contactin, so its secondary structure is multiple homology structures (CH spline structures), in chain, form disulfide linkage.
In fusion rotein of the present invention, between the described solvable fragment of TNFR II and IgG2 Fc fragment, can contain or not contain catenation sequence.Described catenation sequence is the sequence to the effect of not exerting an influence of two albumen normally.As optimal way of the present invention, between the described solvable fragment of TNFR II and IgG2 Fc fragment, do not contain catenation sequence.
TNFR-IgG2/Fc fusion rotein of the present invention, the relatively independent TNFR molecule of its stable antibody-like dimeric structure has effectively extended Half-life in vivo, has obtained the external binding ability higher than the TNF-α of Enbrel simultaneously, has higher biological activity.What is more important, due to IgG2 molecule hinge area sequence and CH2 region sequence, the ability of its conjugated complement and Fc acceptor will far be weaker than IgG1 molecule, has therefore greatly reduced the possibility of induction ADCC and CDC effect.When treatment rheumatoid arthritis or other new indications, TNFR-IgG2/Fc molecular energy of the present invention effectively reduces free TNF-α concentration, has reduced the generation of adverse immune response simultaneously.
According to aminoacid sequence provided by the invention, the art personnel can make fusion rotein of the present invention with various currently known methodss easily.These methods are such as but not limited to recombinant DNA method, synthetic, wait [referring to Murray KM, Dahl SLAnn; Pharmacother 1997 Nov; 31 (11): 1335-8].
Obtaining after the aminoacid sequence of cicada fusion rotein of the present invention, those skilled in the art can obtain the gene order of coding fusion rotein of the present invention easily according to described aminoacid sequence.
As optimal way of the present invention, the encoding gene of fusion rotein of the present invention has the sequence shown in SEQ ID NO:3, adopts this sequence, is particularly suitable for high expression level fusion rotein of the present invention in eukaryotic cell (preferably Chinese hamster ovary celI).
According to nucleotide sequence as herein described, the art personnel can make coding nucleic acid of the present invention with various currently known methodss easily.These methods are such as but not limited to PCR, DNA synthetic etc., and concrete method can be referring to J. Pehanorm Brooker, < < molecular cloning experiment guide > >.As one embodiment of the present invention, the method that can carry out again overlapping extension PCR by salvage nucleotide sequence builds nucleic acid sequence encoding of the present invention.
The present invention also provides a kind of expression vector, comprises the encode sequence of fusion rotein of the present invention and the connected expression regulation sequence of operability with it.Described " operability is connected " or " being operationally connected in " refer to so a kind of situation, and some part of linear DNA sequence can regulate or control the activity of same linear DNA sequence other parts.For example, if the transcribing of promotor control sequence, it is exactly to be operationally connected in encoding sequence so.
Expression vector can adopt commercially available such as but not limited to: pIRES, pDR, pUC18 etc. can be used for the carrier that eukaryotic cell system is expressed.Those skilled in the art can select suitable expression vector according to host cell.As optimal way of the present invention, when when eukaryotic cell is particularly expressed in Chinese hamster ovary celI (Chinese hamster ovary cell), adopt pIRES expression vector.As optimal way of the present invention, when building recombinant vectors, when introducing fusion rotein encoding gene of the present invention in carrier, also introduce Chinese hamster glutamine synthetic enzyme (Glutamine Synthetase, GS) gene, introducing GS gene are conducive to the screening of follow-up positive colony, have weakened the basal transcription level of GS gene by the IRES promotor on carrier.G418 can effectively screen stable transfected cells, and adds the inhibition (MSX) of glutamine synthetase, the preferably stable transfected cells strain of high expression level simultaneously.
According to the restriction enzyme mapping of known unloaded expression vector, those skilled in the art can shear and splicing by Restriction Enzyme according to ordinary method, and the encoding sequence of fusion rotein of the present invention is inserted to suitable restriction site, make recombinant expression vector of the present invention.
The present invention also provides the host cell of expressing fusion rotein of the present invention, wherein contains the encoding sequence of fusion rotein of the present invention.Preferably eukaryotic cell of described host cell, such as but not limited to CHO, COS cell, 293 cells, RSF cell etc.As optimal way of the present invention, described cell is Chinese hamster ovary celI, and it can express fusion rotein of the present invention well, can obtain in conjunction with active good the fusion rotein having good stability.
The present invention also provides a kind of method of preparing fusion rotein of the present invention with recombinant DNA, and its step comprises:
1) provide the nucleotide sequence (as SEQ ID NO:3 sequence) of encoding fusion protein;
2) by 1) nucleotide sequence be inserted into suitable expression vector, obtain recombinant expression vector;
3) by 2) recombinant expression vector import suitable host cell;
4) cultivate under conditions suitable for the expression transformed host cell;
5) collect supernatant liquor, and purified fusion protein product.
Described encoding sequence importing host cell can be adopted to the multiple known technology of this area, such as but not limited to: calcium phosphate precipitation, protoplast fusion, liposome transfection, electroporation, microinjection, reverse transcription method, phage transduction method, alkalimetal ion method.
About cultivation and the expression of host cell can be referring to Olander RM Dev Biol Stand1996; 86:338.Can, by cell and residue in centrifugal removal suspension, collect clear liquid.Can identify by agarose gel electrophoresis technology.
The character that can be basic homogeneous by the above-mentioned fusion protein purification preparing, for example, be single band on SDS-PAGE electrophoresis.For example, when recombinant protein is secreting, expressing, can adopt commercial ultra-filtration membrane to separate described albumen, the such as company such as Millipore, Pellicon product, first will express supernatant and concentrate.Further purifying in addition of the method that concentrated solution can adopt gel chromatography, or adopt the method purifying of ion exchange chromatography.Such as anion-exchange chromatography (DEAE etc.) or cation-exchange chromatography.Gel matrix can be the matrix that agarose, dextran, polymeric amide etc. are usually used in protein purification.Q-or SP-group are comparatively desirable ion-exchange groups.Finally, also available hydroxyapatite adsorption chromatography, metal chelate chromatography, the methods such as hydrophobic interaction chromatography and RPLC (RP-HPLC) are to further refining purifying of above-mentioned purified product.Above-mentioned all purification steps can utilize different combinations, finally make purity of protein reach basic homogeneous.
Can utilize the affinity column of the specific antibody, acceptor or the part that contain described fusion rotein to carry out purifying to the fusion rotein of expressing.According to the characteristic of used affinity column, can utilize conventional method, as the amalgamation polypeptide of method elution of bound on affinity column such as high-salt buffer, change pH.Selectively, the aminoterminal of described fusion rotein or carboxyl terminal also can contain one or more polypeptide fragments, as albumen label.Any suitable label may be used to the present invention.For example, described label can be FLAG, HA, HA1, c-Myc, 6-His or 8-His etc.These labels can be used for fusion rotein to carry out purifying.
As optimal way of the present invention, the inventor has built described antigen-4 fusion protein gene, expresses and purifying acquisition albumen in Chinese hamster ovary celI.First design fusion gene, by gene order by primer synthesize, anneal splicing method, built the recombinant plasmid that has comprised glutamine synthetase (GS) screening-gene and TNFR-IgG2/Fc gene, and utilize the method for electric shock transfection that plasmid is imported to CHO host cell, by the growth pressure of screening of medicaments MSX in substratum, select the cell strain of stable integration goal gene lasting high level expression recombinant protein.Cell strain is large scale culturing in serum-free commercialization substratum, and obtains the higher target protein of purity by protein A affinity chromatography and ion exchange chromatography.The inventor has also utilized immunologic principle and method validation physico-chemical property checking and the external activity of TNFR-IgG2/Fc fusion rotein.By SDS-PAGE, verified the apparent molecular weight of albumen; Utilize ELISA and Western-Blot, proved the structural performance of the TNFR-IgG2/Fc fusion rotein of target protein, and utilize N-end and the order-checking of C-end to confirm the accuracy of protein sequence.The result of external TNF α antagonism in toxicity experiment shows, target protein and TNF alpha molecule have very strong binding ability.
The present invention also provides a kind of composition, and it contains significant quantity (as 0.000001-40wt%; Preferably 0.1-50wt%; Better, 5-40wt%) fusion rotein of the present invention, and pharmaceutically acceptable carrier.
Conventionally, fusion rotein of the present invention can be formulated in to nontoxic, inertia with pharmaceutically acceptable aqueous carrier medium in, wherein pH is about 5-8 conventionally, preferably, pH is about 6-8.
As used herein, term " significant quantity " or " effective dose " refer to and can produce function or amount active and that can be accepted by people and/or animal to people and/or animal.
As used herein, the composition of " pharmaceutically acceptable " is applicable to people and/or Mammals and without excessive bad side reaction (as toxicity, stimulation and transformation reactions), has the material of rational benefit/risk ratio.Term " pharmaceutically acceptable carrier " refers to be used for the treatment of the carrier of agent administration, comprises various vehicle and thinner.
The fusion rotein of the present invention that composition of the present invention contains safe and effective amount and pharmaceutically acceptable carrier.This class carrier comprises (but being not limited to): salt solution, damping fluid, glucose, water, glycerine, ethanol and combination thereof.Conventionally pharmaceutical preparation should match with administering mode, and pharmaceutical composition of the present invention can be made into injection form, for example, with physiological saline or contain glucose and the aqueous solution of other assistant agents is prepared by ordinary method.Described pharmaceutical composition should be manufactured under aseptic condition.The dosage of activeconstituents is treatment significant quantity.Pharmaceutical preparation of the present invention also can be made into sustained release preparation.
The significant quantity of fusion rotein of the present invention can change with severity of the pattern of administration and disease to be treated etc.The selection of preferred significant quantity can be determined (for example, by clinical trial) according to various factors by those of ordinary skills.Described factor includes but not limited to: the pharmacokinetic parameter biological example utilization ratio of described fusion rotein, metabolism, transformation period etc.; The severity of the disease that patient will treat, patient's body weight, patient's immune state, the approach of administration etc.Conventionally, when fusion rotein of the present invention gives with the dosage of about 0.00001mg-50mg/kg the weight of animals (preferably 0.0001mg-10mg/kg the weight of animals) every day, can obtain gratifying effect.For example, by an urgent demand for the treatment of situation, can give the dosage that several times separate every day, or dosage is reduced pari passu.
Fusion rotein of the present invention or the pharmaceutical composition that contains described fusion rotein can be used for the α in conjunction with TNF, thereby can be used for prevention or treatment TNF α abnormal activation relative disease.Described TNF α abnormal activation relative disease includes but not limited to: the illness that a series of TNF-alpha molecules such as rheumatoid arthritis, ankylosing spondylitis, psoriatic, septicemia, asthma, apoplexy, diabetes, Crohn ' s disease cause.
Fusion rotein of the present invention or the pharmaceutical composition that contains described fusion rotein also can with other medicines drug combination.When for prevention or treatment TNF α abnormal activation relative disease, described fusion rotein can general administration, or topical, and the factors such as the kind of concrete visual disease, growth site, progress degree determine.
Major advantage of the present invention is:
(1) fusion rotein of the present invention can specific binding human TNF alpha, effectively reduces free TNF α concentration in human body, the relative disease that treatment TNF α causes; Described fusion rotein has not only retained the activity in conjunction with TNF-α, and possesses the feature of very high TNF α avidity and low side effect (ADCC and CDC).
(2) fusion rotein stability of the present invention is high, and Half-life in vivo is long, has overcome the poor technological deficiency of TNF α acceptor class preparation stability in prior art.
The invention is further illustrated by the following examples, the experimental technique of unreceipted actual conditions in embodiment, conventionally according to normal condition, as Sambrook etc., molecular cloning-laboratory manual, second edition (Molecular Cloning-A Laboratory Manual, Second Edition), press of cold spring harbor laboratory, New York (1989) one books and at (1994) modern molecular biology techniques such as Ausubel, modern technologies (Current Protocols in Molecular Biology, Current Protocol) first, standard method described in two volumes operates.
Carrier, bacterial classification, reagent and source that following examples are used:
PUC18-T, Extaq are Takara company product;
PfuUltra Hotstart DNA Polymerase is Stratagene company product;
Dual-expression vector pIRES is Clontech company product;
DNA ligase, restriction enzyme MluI, XbaI, SalI, NotI are NEB company product;
Plasmid extraction, enzyme are cut product recovery and PCR product reclaims Purelink quick plasmid miniprep kit, the Purelink quick gel extraction kit and the PurelinkPCR purification kit that adopt respectively Invitrogen company.
The acquisition of embodiment 1.hTNFRII-Fc fusion gene expression plasmid
The acquisition of 1.hTNFRII-hIgG2/Fc gene
HTNFRII-hIgG2/Fc is fusion rotein, and its gene order has 1389bp, 463 the amino acid whose albumen of encoding.The wherein solvable fragment in N-terminal hTNFRII extracellular region (maturation protein) gene (705bp/235aa), sequence is as SEQ ID NO:1 (deriving from Genebank NM001066); C-terminal hIgG2-Fc gene (684bp/228aa), sequence SEQ ID NO:2 (deriving from Genebank AK131045).
The oligonucleotide fragment (table 1) of design synthetic gene: fragment length is generally 70 base left and right, approximately has 15 base complementrities between two adjacent oligonucleotide fragments, guarantees that the annealing temperature of each oligonucleotide fragment complementary base is consistent as far as possible.With the synthetic hTNFRII gene of method assembling of overlapping extension PCR.
The hTNFRII-hIgG2/Fc oligonucleotide fragment sequence of table 1 synthetic
The synthetic order of splicing:
(1) by fragment 1 and 2,3 and 4,5 and 6,7 and 8,9 and 10,11 and 12,13 and 14,15 and 16,17 and 18,19 and 20,21 and 22,23 and 24,25 and 26 mix respectively, carry out overlapping extension PCR, obtain 1-2,3-4,5-6,7-8,9-10,11-12,13-14,15-16,17-18,19-20,21-22,23-24,25-26.
PCR reaction system: dNTP 8 μ l; 0.5 μ l PfuUltra enzyme; 10 × damping fluid, 10 μ l; DNA fragmentation 2 × 20 μ l; Water 41.5 μ l, totally 100 μ l.Loop parameter: 94 ℃ × 5min → (94 ℃ × 30s → 60 ℃ × 30s → 2 ℃ × 45s) × 25 → 72 ℃ × 10min.
(2) 1-2,3-4,5-6,7-8,9-10,11-12,13-14,15-16,17-18,19-20,21-22,23-24,25-26 mixes respectively and carries out overlapping extension PCR, obtains 1-4,5-8,9-12,13-16,17-20,21-24 and 25-26.
The each 40 μ l of (1) product are answered in negate, add 0.5 μ l PfuUltra enzyme, 20 μ l water, and loop parameter is constant.
(3) by 1-4,5-8,9-12,13-16,17-20 and 21-24 mix respectively, carry out overlapping extension PCR, obtain 1-8,9-16 and 17-24.
The each 40 μ l of (2) product are answered in negate, add 0.5 μ l PfuUltra enzyme, 20 μ l water, and loop parameter is constant.
(4) by 1-8,9-16,17-24 and 25-26 mix, and carry out overlapping extension PCR and obtain full length gene.
The each 30 μ l of (3) product are answered in negate, add 0.5 μ l PfuUltra enzyme, 10 μ l water, and loop parameter is constant.
(5) use downstream primer the gene of assembling is increased, obtain gene complete sequence.
PCR reaction system: dNTP 8 μ l; 0.5 μ l PfuUltra enzyme; 10 × damping fluid, 10 μ l; DNA fragmentation 10 μ l; The each 2 μ l of primer; Water 67.5 μ l, totally 100 μ l.Loop parameter: 94 ℃ × 5min → (94 ℃ × 30s → 60 ℃ × 30s → 72 ℃ × 60s) × 30 → 72 ℃ × 10min → 4 ℃.
Primer: 5-ATGGCGCCCGTCGCCGTCTGGGCCGCGCT-3 (SEQ ID NO:32)
5-TCATTTACCCGGAGACAGGGAGAGGCTC-3(SEQ ID NO：33)
2. the acquisition of Chinese hamster GS (Glutamine Synthetase) gene
According to GS gene order SEQ ID NO:5 (Genebank X03495) sequence, the oligonucleotide fragment of synthetic this gene, with the synthetic GS gene of method assembling of overlapping extension PCR.
The structure of 3.hTNFRII-Fc fusion gene expression plasmid
(1) SalI and NotI double digestion GS gene and pIRES carrier, reaction system:
37 ℃ of water-bath 2h.
(2) agarose gel electrophoresis, reclaims enzyme and cuts product, adopts Purelink quick gel extraction kit, by test kit description operation.
(3) connect goal gene and carrier, reaction system:
16 ℃ of water-baths are spent the night.
(4) will connect product and transform intestinal bacteria, LB is dull and stereotyped to be cultivated.After the multiple clones of picking cultivate, extract plasmid enzyme restriction next day and identify, select positive colony order-checking to identify, obtain the pIRES-GS recombinant plasmid of GS gene integration at pIRES multiple clone site B.
(5) MluI and XbaI double digestion hIgG-Fc gene and pIRES-GS carrier, reaction system is with (1).
(6) agarose gel electrophoresis, reclaims enzyme and cuts product, adopts Purelink quick gel extraction kit, by test kit description operation.
(7) connect goal gene and carrier, reaction system is with (3).
(8) will connect product and transform intestinal bacteria, LB is dull and stereotyped to be cultivated.After the multiple clones of picking cultivate, extract plasmid enzyme restriction next day and identify (seeing Fig. 1), select positive colony order-checking to identify, obtain the hTNFRII-Fc fusion gene expression plasmid of hTNFRII-hIgG/Fc gene integration at pIRES multiple clone site A.
Expression and the purifying of embodiment 2.hTNFRII-Fc fusion gene
The transfection of 1.CHO cell and screening
Clone and culture condition: FreeStyle CHO-S cell (Invitrogen), culture condition is freestyle medium (Invitrogen), containing 10% dialysis serum (Hyclone), is incubated at 10%CO
2, 37 ℃ of incubators.G418、MSX(Sigma)。
The Lipofectamine that transfection adopts Invitrogen company to produce
tM2000 lipofectamine boxes, to specifications operation.Contrast adopts pIRES empty carrier transfection CHO-S cell.Adopt G418 (200 μ M) and MSX (50 μ g/ml) to combine pressurization screening, within 3 days, change a subculture, pressurize is transferred to 96 orifice plates cultivations by limiting dilution assay after 20 days and screens, and cultivates after 1 week, to get culture supernatant and carry out ELISA mensuration.
4 ℃ of mouse-anti-human T NFR primary antibodie (Sigma) coated elisa plates (Corning) spend the night, after 5% milk sealing, add cells and supernatant, after hatching, add mouse-anti human IgG-Fc/HRP primary antibodie (Sigma), hatch rear TMB reagent colour development, 450nm surveys OD value.With the negative contrast of fresh culture.Utilize ELISA typical curve prepared by TNFR-IgG2Fc standard substance to see Fig. 2.Antibody expression amount result when each cell strain is cultivated in 96 orifice plates is as shown in table 2.
Antibody expression amount when the each cell strain of table 2 is cultivated in 96 orifice plates
| Sample | Observed value (OD450) | Extension rate | Titre (ng/ml) |
| 211p080723-2 | 2.06 | 100 | 13766 |
| 211p080723-3 | 2.181 | 100 | 20714 |
| 211p080723-4 | 2.212 | 100 | 24172 |
| Negative control | 0.046 | 100 | Lower than linearity range |
2. monoclonal enlarged culturing and fusion protein purification
Cell culture condition: be incubated at serum-free EX-CELL 302 (SAFc), cultivate at 10%CO
2, 37 ℃ of incubators.
Select the most much higher clonal expansion to 24 orifice plate of expression level, cultivate and detect protein expression after 3 days, select to express high clone and enter next round amplification; 6 clonal expansions that after many wheel amplifications, selection expression level is the highest are to T75 culturing bottle, by the preservation of cell label.Clone the highest expression level is inoculated in 1L rolling bottle, with containing EX-CELL 302 culture medium culturing of 5% calf serum, when cell covers with bottle wall, is changed to serum free medium, receive every other day liquid, receive liquid 3-5 time continuously.
Utilize the specific adsorption effect of albumin A to IgG, adopt separating medium rProtein A Sepharose4Fast Flow (Amersham Biosciences) and Q Sepharose FF (Amersham Biosciences) to carry out affinity chromatography and ion exchange chromatography, purifying expressing protein, as Fig. 3.Working method is referring to the description of product.After purifying, with ultraviolet spectrophotometer, measure A260 and A280 value.Protein quantification formula: protein content (mg/ml)=OD280 value × 1.45-OD260 value × 0.74.
The active detection of physics and chemistry of embodiment 3.hTNFRII-Fc fusion rotein
1.SDS-PAGE protein electrophoresis and Western trace
Get 20 μ l purifying proteins and carry out SDS-PAGE electrophoresis, electrophoretic separation gum concentration chooses 10%, and result is as Fig. 4.Concrete operation step refers to < < molecular cloning > > second edition and protein electrophorese experimental technique.
Sample shifts in nitrocellulose filter after SDS-PAGE electrophoretic separation.Binding antibody after the 5% milk soln sealing containing 0.5% tween for film, primary antibodie is mouse anti human TNFR (Sigma), two resist the goat anti-mouse IgG (Calbiochem) for horseradish peroxidase-labeled, TMB colour developing after PBS/0.5%Tween washes 3 times, and result is as Fig. 5.
2.L929 cytotoxicity neutralization test
Material and reagent: L929 (ATCC), culture condition is RPMI 1640 (Sigma), containing 10% foetal calf serum (Invitrogen), is incubated at 5%CO
2, 37 ℃ of incubators.TNF α (Sigma) is dissolved to 10000u/ml with RPMI 1640, and packing 0.1ml/ props up.Dactinomycin is dissolved to 1mg/ml with RPMI 1640.TNFR-Fc reference material (standard): Enbrel.The biochemical CCK-8 cell counting of colleague test kit.
(1) the L929 cell of taking the logarithm vegetative period, digestion counting, adds 96 orifice plates, 3 × 10
5/ ml × 0.1ml/ hole, overnight incubation.
(2) get 12ml RPMI 1640 next day and dilute actinomycin mother liquor 0.12ml to final concentration 10 μ g/ml, get 2ml (culture medium A) in contrast, it is 10 μ g/ml (substratum B) that all the other 10ml add TNF α mother liquor to concentration.
(3) get the TNFR-Fc reference material of a packing, dissolve and be diluted to concentration 256ng/ml with 1ml substratum B.
(4) according to the protein quantification of testing sample, be dissolved to 0.1mg/ml with aseptic PBS, then in the aseptic centrifuge tube of 1.5ml, with substratum B, carry out gradient dilution.
(5) respectively in aseptic centrifuge tube with the diluent of the continuous doubling dilution reference material of substratum or sample.The volume of each diluent is 0.4ml.
(6) use nutrient solution A as negative control, nutrient solution B is as positive control.
(7) in the orifice plate of inoculating cell, add the sample, standard substance and the positive and negative contrast liquid that have diluted, 0.1ml/ hole, each measurement point is established 3 multiple holes.5%CO2,37 ℃ of incubators continue to cultivate.
(8) hatch after 5-10h, discard substratum, add the freshly prepared RPMI1640 containing 10%CCK-8,0.1ml/ hole, continues to hatch 15min.
(9) add 10 μ l/ hole 10%SDS termination reactions, by microplate reader, measure its value A450, reference is A650.
From measurement result (as Fig. 6), can find out, TNF α in vitro toxicity in conjunction with experiment in, by multiple cell strain (211P80723-2,211P80723-3, sample 211P80723-4) obtaining all has very strong TNF α binding ability, and it all will be significantly higher than the Enbrel albumen of same dosage to the antagonistic ability of TNF α, be respectively 2.66 of Enbrel activity, 1.26 with 1.26 times, as table 3.
Table 3TNF α in vitro toxicity is in conjunction with the conclusion of experiment
| Sample title | Enbrel | 211P80723-2 | 211P080723-3 | 211P080723-4 |
| (μ g/ μ l) for EC50 value | 0.0258 | 0.00971 | 0.0205 | 0.0205 |
| Relation conefficient (R 2) | 0.9973 | 0.9992 | 0.9963 | 0.9977 |
| EC50 work reference material/EC50 sample | / | 2.657 | 1.259 | 1.259 |
The pharmacokinetic of embodiment 4.hTNFR II-Fc fusion rotein in rat body
504 of rats, be divided at random 6 groups, self-control sample is hTNFR II-IgG2/Fc fusion rotein, control sample is the commercially available Enbrel of Wyeth company, respectively by 0.5, 3, 18mg/kg subcutaneous injection administration, after administration 1, 2, 4, 8, 12, 24, 28, 32, 36, 48, 72, 96, 120, 14 time point heart blood samplings such as 144h, 6 animal (3 ♂ of each time point, 3 ♀), centrifugal, divide and get serum, with the content of kit measurement serum hTNFR II, calculate the Plasma Concentration of different time, application pharmacokinetics and 3P87 software carry out c-t fitting of a curve to Plasma Concentration (c) and time (t) data, and calculate relevant pharmacokinetic parameter.
Enzyme linked immunosorbent assay is measured different time rhTNFR Plasma Concentration, during drug level-time data process of fitting treatment subcutaneous injection hTNFR II-IgG2/Fc medicine, curve meets the feature of the non-vascular drug delivery of one compartment model, and 0.5,3, the main result parameter of 18mg/kg Three doses is as shown in table 4.Under Isodose, hTNFR II-IgG2/Fc has the significantly higher metabolism cycle than Enbrel, and its Half-life in vivo is higher by 25.64% than Enbrel.
Table 4
T
1/2 (ka)represent: absorption halftime;
T
1/2 (ke)represent: eliminate the transformation period;
Tpeak represents: peak time.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted separately as a reference.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Sequence table
The glad profit of <110> (Shanghai) Bioceuticals Inc.
<120> TNFR-Fc fusion rotein and uses thereof
<130>085756
<160>33
<170>PatentIn version 3.3
<210>1
<211>235
<212>PRT
<213> homo sapiens (Homo Sapiens)
<400>1
Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu Pro Gly Ser
1 5 10 15
Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln Met Cys Cys
20 25 30
Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys Thr Lys Thr
35 40 45
Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr Tyr Thr Gln Leu
50 55 60
Trp Asn Trp Val Pro Glu Cys Leu Ser Cys Gly Ser Arg Cys Ser Ser
65 70 75 80
Asp Gln Val Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn Arg Ile Cys
85 90 95
Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln Glu Gly Cys
100 105 110
Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe Gly Val Ala
115 120 125
Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro Cys Ala Pro
130 135 140
Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys Arg Pro His
145 150 155 160
Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser Met Asp Ala
165 170 175
Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala Pro Gly Ala Val
180 185 190
His Leu Pro Gln Pro Val Ser Thr Arg Ser Gln His Thr Gln Pro Thr
195 200 205
Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu Pro Met Gly
210 215 220
Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp
225 230 235
<210>2
<211>228
<212>PRT
<213> homo sapiens (Homo Sapiens)
<400>2
Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val
1 5 10 15
Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
20 25 30
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
35 40 45
His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
50 55 60
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
65 70 75 80
Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn
85 90 95
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro
100 105 110
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln
115 120 125
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
130 135 140
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
145 150 155 160
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
165 170 175
Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
180 185 190
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
195 200 205
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
210 215 220
Ser Pro Gly Lys
225
<210>3
<211>1392
<212>DNA
<213> artificial sequence
<220>
<221>misc_feature
<223> polynucleotide
<400>3
ttgcccgccc aggtggcatt tacaccctac gccccggagc ccgggagcac atgccggctc 60
agagaatact atgaccagac agctcagatg tgctgcagca aatgctcgcc gggccaacat 120
gcaaaagtct tctgtaccaa gacctcggac accgtgtgtg actcctgtga ggacagcaca 180
tacacccagc tctggaactg ggttcccgag tgcttgagct gtggctcccg ctgtagctct 240
gaccaggtgg aaactcaagc ctgcactcgg gaacagaacc gcatctgcac ctgcaggccc 300
ggctggtact gcgcgctgag caagcaggag gggtgccggc tgtgcgcgcc gctgcgcaag 360
tgccgcccgg gcttcggcgt ggccagacca ggaactgaaa catcagacgt ggtgtgcaag 420
ccctgtgccc cggggacgtt ctccaacacg acttcatcca cggatatttg caggccccac 480
cagatctgta acgtggtggc catccctggg aatgcaagca tggatgcagt ctgcacgtcc 540
acgtccccca cccggagtat ggccccaggg gcagtacact taccccagcc agtgtccaca 600
cgatcccaac acacgcagcc aactccagaa cccagcactg ctccaagcac ctccttcctg 660
ctcccaatgg gccccagccc cccagctgaa gggagcactg gcgacgagcg caaatgttgt 720
gtcgagtgcc caccgtgccc agcaccacct gtggcaggac cgtcagtctt cctcttcccc 780
ccaaaaccca aggacaccct catgatctcc cggacccctg aggtcacgtg cgtggtggtg 840
gacgtgagcc acgaagaccc cgaggtccag ttcaactggt acgtggacgg cgtggaggtg 900
cataatgcca agacaaagcc acgggaggag cagttcaaca gcacgttccg tgtggtcagc 960
gtcctcaccg tcgtgcacca ggactggctg aacggcaagg agtacaagtg caaggtctcc 1020
aacaaaggcc tcccagcccc catcgagaaa accatctcca aaaccaaagg gcagccccga 1080
gaaccacagg tgtacaccct gcccccatcc cgggaggaga tgaccaagaa ccaggtcagc 1140
ctgacctgcc tggtcaaagg cttctacccc agcgacatcg ccgtggagtg ggagagcaat 1200
gggcagccgg agaacaacta caagaccacg cctcccatgc tggactccga cggctccttc 1260
ttcctctaca gcaagctcac cgtggacaag agcaggtggc agcaggggaa cgtcttctca 1320
tgctccgtga tgcatgaggc tctgcacaac cactacacgc agaagagcct ctccctgtct 1380
ccgggtaaat ga 1392
<210>4
<211>373
<212>PRT
<213> hamster (Cricetidae)
<400>4
Met Ala Thr Ser Ala Ser Ser His Leu Asn Lys Asn Ile Lys Gln Met
1 5 10 15
Tyr Leu Cys Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp
20 25 30
Val Asp Gly Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp
35 40 45
Cys Glu Pro Lys Cys Val Glu Glu Leu Pro Glu Trp Asn Phe Asp Gly
50 55 60
Ser Ser Thr Phe Gln Ser Glu Gly Ser Asn Ser Asp Met Tyr Leu Ser
65 70 75 80
Pro Val Ala Met Phe Arg Asp Pro Phe Arg Arg Asp Pro Asn Lys Leu
85 90 95
Val Phe Cys Glu Val Phe Lys Tyr Asn Arg Lys Pro Ala Glu Thr Asn
100 105 110
Leu Arg His Ser Cys Lys Arg Ile Met Asp Met Val Ser Asn Gln His
115 120 125
Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Met Gly Thr Asp Gly
130 135 140
His Pro Phe Gly Trp Pro Ser Asn Gly Phe Pro Gly Pro Gln Gly Pro
145 150 155 160
Tyr Tyr Cys Gly Val Gly Ala Asp Lys Ala Tyr Gly Arg Asp Ile Val
165 170 175
Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile Thr Gly
180 185 190
Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe Gln Ile Gly Pro
195 200 205
Cys Glu Gly Ile Arg Met Gly Asp His Leu Trp Val Ala Arg Phe Ile
210 215 220
Leu His Arg Val Cys Glu Asp Phe Gly Val Ile Ala Thr Phe Asp Pro
225 230 235 240
Lys Pro Ile Pro Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe
245 250 255
Ser Thr Lys Ala Met Arg Glu Glu Asn Gly Leu Lys His Ile Glu Glu
260 265 270
Ala Ile Glu Lys Leu Ser Lys Arg His Arg Tyr His Ile Arg Ala Tyr
275 280 285
Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Gly Leu Thr Gly Phe His
290 295 300
Glu Thr Ser Asn Ile Asn Asp Phe Ser Ala Gly Val Ala Asn Arg Ser
305 310 315 320
Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln Glu Lys Lys Gly Tyr
325 330 335
Phe Glu Asp Arg Arg Pro Ser Ala Asn Cys Asp Pro Phe Ala Val Thr
340 345 350
Glu Ala Ile Val Arg Thr Cys Leu Leu Asn Glu Thr Gly Asp Glu Pro
355 360 365
Phe Gln Tyr Lys Asn
370
<210>5
<211>1122
<212>DNA
<213> hamster (Cricetidae)
<400>5
atggccacct cagcaagttc ccacttgaac aaaaacatca agcaaatgta cttgtgcctg 60
ccccagggtg agaaagtcca agccatgtat atctgggttg atggtactgg agaaggactg 120
cgctgcaaaa cccgcaccct ggactgtgag cccaagtgtg tagaagagtt acctgagtgg 180
aattttgatg gctctagtac ctttcagtct gagggctcca acagtgacat gtatctcagc 240
cctgttgcca tgtttcggga ccccttccgc agagatccca acaagctggt gttctgtgaa 300
gttttcaagt acaaccggaa gcctgcagag accaatttaa ggcactcgtg taaacggata 360
atggacatgg tgagcaacca gcacccctgg tttggaatgg aacaggagta tactctgatg 420
ggaacagatg ggcacccttt tggttggcct tccaatggct ttcctgggcc ccaaggtccg 480
tattactgtg gtgtgggcgc agacaaagcc tatggcaggg atatcgtgga ggctcactac 540
cgcgcctgct tgtatgctgg ggtcaagatt acaggaacaa atgctgaggt catgcctgcc 600
cagtgggaat tccaaatagg accctgtgaa ggaatccgca tgggagatca tctctgggtg 660
gcccgtttca tcttgcatcg agtatgtgaa gactttgggg taatagcaac ctttgacccc 720
aagcccattc ctgggaactg gaatggtgca ggctgccata ccaactttag caccaaggcc 780
atgcgggagg agaatggtct gaagcacatc gaggaggcca tcgagaaact aagcaagcgg 840
caccggtacc acattcgagc ctacgatccc aaggggggcc tggacaatgc ccgtggtctg 900
actgggttcc acgaaacgtc caacatcaac gacttttctg ctggtgtcgc caatcgcagt 960
gccagcatcc gcattccccg gactgtcggc caggagaaga aaggttactt tgaagaccgc 1020
cgcccctctg ccaattgtga cccctttgca gtgacagaag ccatcgtccg cacatgcctt 1080
ctcaatgaga ctggcgacga gcccttccaa tacaaaaact aa 1122
<210>6
<211>70
<212>DNA
<213> oligonucleotide
<400>6
atggcgcccg tcgccgtctg ggccgcgctg gccgtcggac tggagctctg ggctgcggcg 60
cacgccttgc 70
<210>7
<211>70
<212>DNA
<213> oligonucleotide
<400>7
gccgcgtgcg gaacgggcgg gtccaccgta aatgtgggat gcggggcctc gggccctcgt 60
gtacggccga 70
<210>8
<211>70
<212>DNA
<213> oligonucleotide
<400>8
gagcacatgc cggctcagag aatactatga ccagacagct cagatgtgct gcagcaaatg 60
ctcgccgggc 70
<210>9
<211>70
<212>DNA
<213> oligonucleotide
<400>9
tttacgagcg gcccggttgt acgttttcag aagacatggt tctggagcct gtggcacaca 60
ctgaggacac 70
<210>10
<211>70
<212>DNA
<213> oligonucleotide
<400>10
tgtgtgactc ctgtgaggac agcacataca cccagctctg gaactgggtt cccgagtgct 60
tgagctgtgg 70
<210>11
<211>70
<212>DNA
<213> oligonucleotide
<400>11
cacgaactcg acaccgaggg cgacatcgag actggtccac ctttgagttc ggacgtgagc 60
ccttgtcttg 70
<210>12
<211>70
<212>DNA
<213> oligonucleotide
<400>12
actcgggaac agaaccgcat ctgcacctgc aggcccggct ggtactgcgc gctgagcaag 60
caggaggggt 70
<210>13
<211>70
<212>DNA
<213> oligonucleotide
<400>13
cgttcgtcct ccccacggcc gacacgcgcg gcgacgcgtt cacggcgggc ccgaagccgc 60
accggtctgg 70
<210>14
<211>70
<212>DNA
<213> oligonucleotide
<400>14
cggcgtggcc agaccaggaa ctgaaacatc agacgtggtg tgcaagccct gtgccccggg 60
gacgttctcc 70
<210>15
<211>70
<212>DNA
<213> oligonucleotide
<400>15
ggcccctgca agaggttgtg ctgaagtagg tgcctataaa cgtccggggt ggtctagaca 60
ttgcaccacc 70
<210>16
<211>70
<212>DNA
<213> oligonucleotide
<400>16
tctgtaacgt ggtggccatc cctgggaatg caagcatgga tgcagtctgc acgtccacgt 60
cccccacccg 70
<210>17
<211>70
<212>DNA
<213> oligonucleotide
<400>17
gtgcaggggg tgggcctcat accggggtcc ccgtcatgtg aatggggtcg gtcacaggtg 60
tgctagggtt 70
<210>18
<211>70
<212>DNA
<213> oligonucleotide
<400>18
tccacacgat cccaacacac gcagccaact ccagaaccca gcactgctcc aagcacctcc 60
ttcctgctcc 70
<210>19
<211>70
<212>DNA
<213> oligonucleotide
<400>19
ggaggaagga cgagggttac ccggggtcgg ggggtcgact tccctcgtga ccgctgctcg 60
cgtttacaac 70
<210>20
<211>70
<212>DNA
<213> oligonucleotide
<400>20
cgagcgcaaa tgttgtgtcg agtgcccacc gtgcccagca ccacctgtgg caggaccgtc 60
agtcttcctc 70
<210>21
<211>70
<212>DNA
<213> oligonucleotide
<400>21
ggcagtcaga aggagaaggg gggttttggg ttcctgtggg agtactagag ggcctgggga 60
ctccagtgca 70
<210>22
<211>70
<212>DNA
<213> oligonucleotide
<400>22
cccctgaggt cacgtgcgtg gtggtggacg tgagccacga agaccccgag gtccagttca 60
actggtacgt 70
<210>23
<211>70
<212>DNA
<213> oligonucleotide
<400>23
caagttgacc atgcacctgc cgcacctcca cgtattacgg ttctgtttcg gtgccctcct 60
cgtcaagttg 70
<210>24
<211>70
<212>DNA
<213> oligonucleotide
<400>24
gaggagcagt tcaacagcac gttccgtgtg gtcagcgtcc tcaccgtcgt gcaccaggac 60
tggctgaacg 70
<210>25
<211>70
<212>DNA
<213> oligonucleotide
<400>25
tcctgaccga cttgccgttc ctcatgttca cgttccagag gttgtttccg gagggtcggg 60
ggtagctctt 70
<210>26
<211>70
<212>DNA
<213> oligonucleotide
<400>26
agcccccatc gagaaaacca tctccaaaac caaagggcag ccccgagaac cacaggtgta 60
caccctgccc 70
<210>27
<211>70
<212>DNA
<213> oligonucleotide
<400>27
cacatgtggg acgggggtag ggccctcctc tactggttct tggtccagtc ggactggacg 60
gaccagtttc 70
<210>28
<211>70
<212>DNA
<213> oligonucleotide
<400>28
cctgcctggt caaaggcttc taccccagcg acatcgccgt ggagtgggag agcaatgggc 60
agccggagaa 70
<210>29
<211>70
<212>DNA
<213> oligonucleotide
<400>29
acccgtcggc ctcttgttga tgttctggtg cggagggtac gacctgaggc tgccgaggaa 60
gaaggagatg 70
<210>30
<211>70
<212>DNA
<213> oligonucleotide
<400>30
tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 60
ttctcatgct 70
<210>31
<211>83
<212>DNA
<213> oligonucleotide
<400>31
tgcagaagag tacgaggcac tacgtactcc gagacgtgtt ggtgatgtgc gtcttctcgg 60
agagggacag aggcccattt act 83
<210>32
<211>29
<212>DNA
<213> primer
<400>32
atggcgcccg tcgccgtctg ggccgcgct 29
<210>33
<211>28
<212>DNA
<213> primer
<400>33
tcatttaccc ggagacaggg agaggctc 28
Claims (12)
1. a fusion rotein, is characterized in that, the aminoterminal of described fusion rotein is the solvable fragment of human tumor necrosis factor II receptor; Carboxyl terminal is the Fc fragment of human normal immunoglobulin 2; The aminoacid sequence of the described solvable fragment of human tumor necrosis factor II receptor is as shown in SEQ ID NO:1; The aminoacid sequence of the Fc fragment of described human normal immunoglobulin 2 is as shown in SEQ ID NO:2.
2. a nucleic acid molecule, is characterized in that, described nucleic acid molecule encoding fusion rotein claimed in claim 1.
3. nucleic acid molecule as claimed in claim 2, is characterized in that, the nucleotide sequence of described nucleic acid molecule is as shown in SEQ ID NO:3.
4. a carrier, is characterized in that, it contains nucleic acid molecule claimed in claim 3.
5. carrier as claimed in claim 4, is characterized in that, is also connected with operably the encoding gene of glutamine synthetase in described carrier.
6. carrier as claimed in claim 5, is characterized in that, the aminoacid sequence of described glutamine synthetase is as shown in SEQ ID NO:4.
7. carrier as claimed in claim 5, is characterized in that, described carrier is pIRES two-cistron expression vector.
8. a genetically engineered cell, is characterized in that,
Described cell contains the arbitrary described carrier of claim 4-7; Or
In described cellular genome, be integrated with the nucleic acid molecule described in claim 2 or 3.
9. a method that produces fusion rotein claimed in claim 1, is characterized in that, described method comprises: being applicable to expressing under the condition of described fusion rotein, cultivate host cell claimed in claim 8, express and isolate described fusion rotein.
10. the purposes of fusion rotein claimed in claim 1 in the composition of preparing specific binding tumor necrosis factor alpha.
11. purposes as claimed in claim 10, is characterized in that, described composition is for prevention or treatment tumor necrosis factor alpha abnormal activation relative disease.
12. a composition for specific binding tumor necrosis factor alpha, is characterized in that, described composition contains:
(i) fusion rotein claimed in claim 1 of significant quantity; With
(ii) pharmaceutically acceptable carrier.
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| NZ629309A (en) * | 2012-02-22 | 2016-03-31 | Nvip Pty Ltd | Tumour necrosis factor receptor fusion proteins and methods of using the same |
| CN111228225B (en) * | 2018-11-28 | 2022-08-26 | 鲁南制药集团股份有限公司 | Recombinant human tumor necrosis factor receptor-Fc fusion protein freeze-dried preparation |
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| CN1524092A (en) * | 2001-07-26 | 2004-08-25 | ÷�¿�˹����ʽ���� | Concatameric immunoadhesion |
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