CN101747440A - TNFR-Fc fusion protein and usage thereof - Google Patents

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

TNFR-Fc fusion protein and application thereof

Technical Field

The invention belongs to the field of molecular biology, and relates to a novel recombinant TNFR-Fc fusion protein and application thereof.

Background

Human tumor necrosis factor (hTNF-alpha) is a cytokine mainly produced by an activated monocyte/macrophage, and is a cytokine with various biological effects, and early studies find that TNF-alpha can induce tumor cell necrosis or apoptosis, but later, TNF-alpha is a main cytokine for mediating inflammatory response, is also an important factor for causing fever and septic shock, and plays an important role in heart failure, transplant rejection and autoimmune diseases. An appropriate amount of TNF-alpha can activate the immune system, enhance immunity, and play a key role in the host defense system against microbial invasion and inhibition of tumor production. However, excessive TNF- α expression can produce a variety of pathological lesions with other inflammatory factors. Thus, TNF- α acts as a double-edged sword in vivo.

TNF-alpha levels significantly higher than normal have been demonstrated in a variety of disease studies and have been implicated in its possible pathogenesis (Feldman M, et al, Ann Rev Immunol 1996; 14: 397). As TNF-alpha is involved in the process of occurrence and development of various diseases, plays a key role in certain diseases and is combined with other factors in certain diseases; therefore, blocking the effects of TNF-alpha at various levels has the potential to produce therapeutic effects on TNF-alpha related diseases. At present, some diseases treated by taking TNF-alpha as a target are approved to be clinically used, expected effects are obtained, and some diseases are undergoing clinical experiments.

Two different TNFRs, TNFRI with molecular weight of 55KD (CD120 alpha) and TNFRII with molecular weight of 75KD (CD120 beta), are present on the cell surface. Human TNF-alpha has a high affinity for TNFR with affinity constants of 1.23 + -0.23 nM and 0.36 + -0.13 nM for TNFRI and TNFRII, respectively. Both TNFR types have shed off their extracellular domain and still retain TNF- α binding activity. The extracellular domain of the receptor is linked to the Fc region of the antibody, and the resulting fusion protein can bind to TNF- α with increased stability, and can form a dimer via the Fc region with greater affinity for TNF- α than naturally occurring monomeric receptors.

In the prior art, the company Immunex in the United states developed a commercial TNFRII-IgG1/Fc fusion protein, named Etanercept, under the trade name Enbrel, which has been approved by the FDA for the treatment of rheumatoid arthritis, ankylosing spondylitis and the inhibition of bone and joint damage in patients with psoriasis. However, many marketed TNF molecule antagonist drugs, including Enbrel, have certain side effects due to their potential to induce ADCC and CDC in vivo (Tracey D, et al, Pharmacol Ther.2008; 117 (2): 244-79).

However, the preparations that bind TNF α found so far have the disadvantages of low binding ability, low bioactivity and short effective duration in vivo, and thus there is a need in the art for further research on improved drugs effective in improving the therapeutic effects of the drugs.

Disclosure of Invention

The invention aims to provide a fusion protein, wherein the amino terminal of the fusion protein is a soluble fragment of a human tumor necrosis factor II type receptor (TNFR II); the carboxy terminus is the Fc fragment of human IgG 2.

The invention also aims to provide the application and the composition of the fusion protein.

In a first aspect of the present invention, there is provided a fusion protein, wherein the amino terminus of the fusion protein is a human tumor necrosis factor type II receptor (TNFR II) soluble fragment; at the carboxy terminus is the Fc fragment of human immunoglobulin 2(IgG 2).

In another preferred embodiment, the fusion protein has a half-life in vivo of greater than 48 hours.

In another preferred embodiment, the human tumor necrosis factor type II receptor has the amino acid sequence of SEQ ID NO: 1. Preferably, the amino acid sequence of the soluble fragment of human type II TNF α receptor is as set forth in SEQ ID NO: 1 is shown.

In another preferred embodiment, the Fc fragment of human IgG2 has the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof. Preferably, the amino acid sequence of the Fc fragment of human IgG2 is as shown in SEQ ID NO: 2, respectively.

In a second aspect of the invention, there is provided a nucleic acid molecule encoding the fusion protein.

In another preferred embodiment, the nucleic acid molecule has the sequence of SEQ ID NO: 3.

In a third aspect of the invention, there is provided a vector comprising said nucleic acid molecule.

In another preferred embodiment, the vector further comprises a gene encoding Glutamine Synthetase (GS) operably linked thereto.

In another preferred embodiment, the amino acid sequence of the glutamine synthetase is as shown in SEQ ID NO: 4, respectively.

In another preferred embodiment, the gene sequence of the glutamine synthetase is shown in SEQ ID NO: 5 (or sequence information such as Genebank X03495).

In another preferred embodiment, the vector is a pIRES bicistronic expression vector.

In another preferred embodiment, the gene sequence encoding glutamine synthetase is located at the multiple cloning site B of the expression vector; the above-mentioned nucleic acid molecule is positioned in the multiple cloning site A of said expression vector.

In a fourth aspect of the invention, there is provided a genetically engineered cell,

said cell comprising said vector;

or said nucleic acid molecule is integrated into the genome of said cell.

In a fifth aspect of the invention, there is provided a method of producing said fusion protein, said method comprising: culturing said host cell under conditions suitable for expression of said fusion protein, expressing and isolating said fusion protein.

In the sixth aspect of the invention, the use of the fusion protein is provided for preparing a composition specifically binding to tumor necrosis factor alpha.

In another preferred embodiment, the composition is used for preventing or treating diseases related to the abnormal activation of tumor necrosis factor alpha.

In another preferred embodiment, the diseases associated with the abnormal activation of TNF-alpha include: rheumatoid arthritis, ankylosing spondylitis, psoriasis, sepsis, asthma, stroke, diabetes, Crohn's disease.

In a seventh aspect of the present invention, there is provided a composition that specifically binds to tumor necrosis factor α, said composition comprising:

(i) an effective amount of said fusion protein; and

(ii) a pharmaceutically acceptable carrier.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1 shows the results of restriction enzyme analysis of recombinant plasmid. Wherein, Lane 1 is DL250bp Marker; lane 2 is PCR verified recombinant plasmid; lane 3 is a double restriction enzyme identified recombinant plasmid; lane 4 shows the markers DL 500-15,000 bp.

FIG. 2 ELISA standard curve prepared using TNFR-IgG2Fc standard.

FIG. 3 shows the purification of expressed proteins by affinity chromatography and ion exchange chromatography using separation media rProtein A Sepharose4Fast Flow and Q Sepharose FF.

FIG. 4 SDS-PAGE detection of purified proteins. Wherein,

lane 1. protein labeling; lane 2. non-reducing sample (211p 080723-2);

lane 3. non-reducing sample (211p 080723-3); lane 4. non-reducing sample (211p 080723-4);

lane 5. non-reducing sample (Enbrel); lane 6. protein labeling;

lane 7. reduction sample (Enbrel); lane 8. reduced sample (211p 080723-2);

lane 9. reduced sample (211p 080723-3); lane 10. reduction sample (211p 080723-4).

FIG. 5 Western blot detection of purified protein. Wherein,

lane 1. protein labeling; lane 2. non-reducing sample (211p 080723-2);

lane 3. non-reducing sample (211p 080723-3); lane 4. non-reducing sample (211p 080723-4);

lane 5. non-reducing sample (Enbrel); lane 6. protein labeling;

lane 7. reduction sample (Enbrel); lane 8. reduced sample (211p 080723-2);

lane 9. reduced sample (211p 080723-3); lane 10. reduction sample (211p 080723-4).

Figure 6 original results of TNF α in vitro toxicity binding experiments.

Detailed Description

The present inventors have made intensive studies and have unexpectedly found that a fusion protein obtained by fusing a soluble fragment of human tumor necrosis factor type II receptor (TNFRII) with an Fc fragment of human IgG2 has an extremely excellent TNF α -binding effect, and is excellent in stability, long in half-life, and low in side effects.

As used herein, TNFR, TNFR II and hTNFR II are used interchangeably and refer to human tumor necrosis factor II type receptors, unless otherwise indicated.

As used herein, unless otherwise specified, IgG2Fc or hIgG/Fc refers to the Fc fragment of human immunoglobulin 2, which is different from the Fc fragment of human immunoglobulin 1 (IgG1 Fc).

As used herein, the terms "comprising," "having," or "including" include "comprising," "consisting essentially of, and" consisting of,. once; "consists essentially of," and "consists of" belong to the subordinate concepts of "containing," having, "or" including.

As used herein, unless otherwise indicated, the fusion protein is an isolated protein, unrelated to other proteins, polypeptides or molecules, purified product of recombinant host cell culture or as a purified extract.

The invention provides a fusion protein, which comprises a soluble fragment of a human soluble tumor necrosis factor II type receptor and an Fc fragment (IgG2Fc) of human immunoglobulin 2. The fusion protein can be used for preparing a composition specifically binding to tumor necrosis factor alpha. The fusion protein is abbreviated as "TNFR-IgG 2/Fc" or "hTNFR II-IgG 2/Fc".

In the fusion protein, the amino terminal is a TNFR II soluble fragment; the carboxy terminus is an IgG2Fc fragment, which has a hinge region, CH2 and CH3 regions.

The TNFR-IgG2/Fc fusion protein has a relative molecular weight of 150 kilodaltons, wherein the amino acid residue accounts for about 100 kilodaltons, and the rest is a sugar chain. Each peptide chain contains 3N glycosylation sites, and 2 polypeptide chains can form a disulfide bond through cysteine residues to form an isodimer. Since TNFR II also belongs to a member of the immunoglobulin superfamily, its secondary structure is a multiple of homologous structures (CH-like structures), within which disulfide bonds are formed.

In the fusion protein of the present invention, the connecting sequence may or may not be included between the soluble fragment of TNFR II and the IgG2Fc fragment. The linker sequence is generally a sequence that does not affect both proteins. In a preferred embodiment of the present invention, the TNFR II soluble fragment and the IgG2Fc fragment do not contain a linker sequence.

The stable antibody-like dimer structure of the TNFR-IgG2/Fc fusion protein effectively prolongs the half-life in vivo compared with a single TNFR molecule, and simultaneously obtains the in vitro binding capacity of TNF-alpha higher than that of Enbrel, and has higher biological activity. More importantly, due to the characteristics of the hinge region sequence and the CH2 region sequence of the IgG2 molecule, the capability of combining complement and Fc receptor is far weaker than that of the IgG1 molecule, thereby greatly reducing the possibility of inducing ADCC and CDC effects. When used for treating rheumatoid arthritis or other new indications, the TNFR-IgG2/Fc molecule can effectively reduce the concentration of free TNF-alpha and reduce the occurrence of adverse immune response.

Based on the amino acid sequences provided by the present invention, the fusion protein of the present invention can be conveniently prepared by various known methods by those skilled in the art. Such methods are for example but not limited to: recombinant DNA methods, artificial synthesis, etc. [ see Murray KM, Dahl SLAnn; pharmacother 1997 Nov; 31(11): 1335-8].

After the amino acid sequence of the fusion protein of the present invention is known, the skilled person can conveniently obtain the gene sequence encoding the fusion protein of the present invention based on the amino acid sequence.

As a preferred mode of the present invention, the encoding gene of the fusion protein of the present invention has the sequence of SEQ ID NO: 3, and is particularly suitable for high expression of the fusion protein in eukaryotic cells (preferably CHO cells).

The coding nucleic acids of the invention can be readily prepared by one of skill in the art using a variety of known methods based on the nucleotide sequences described herein. Such methods are for example but not limited to: PCR, DNA synthesis, etc., and specific methods can be found in sambrook, molecular cloning guidelines. As an embodiment of the present invention, the coding nucleic acid sequence of the present invention can be constructed by a method of synthesizing nucleotide sequences by segmentation and then performing overlap extension PCR.

The invention also provides an expression vector comprising a sequence encoding the fusion protein of the invention and an expression control sequence operably linked thereto. The term "operably linked" or "operably linked" refers to the condition wherein certain portions of a linear DNA sequence are capable of modulating or controlling the activity of other portions of the same linear DNA sequence. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence.

The expression vector may be any one commercially available, for example, but not limited to: pIRES, pDR, pUC18 and the like can be used as vectors for eukaryotic cell system expression. One skilled in the art can select an appropriate expression vector depending on the host cell. In a preferred embodiment of the present invention, pIRES expression vectors are used when expression is carried out in eukaryotic cells, particularly CHO cells (Chinese hamster ovary cells). As a preferred mode of the invention, when a recombinant vector is constructed, Chinese hamster Glutamine Synthetase (GS) gene is introduced into the vector, and GS gene is introduced to facilitate the screening of subsequent positive clones while introducing the fusion protein coding gene of the invention, so that the basic transcription level of the GS gene is weakened by an IRES promoter on the vector. G418 can effectively screen stably transfected cells, and simultaneously, a glutamine synthetase inhibitor (MSX) is added, so that a stably transfected cell strain with high expression can be preferred.

According to the enzyme cutting map of the known no-load expression vector, the technical personnel can insert the coding sequence of the fusion protein into a proper restriction site by restriction enzyme cutting and splicing according to the conventional method to prepare the recombinant expression vector.

The invention also provides host cells expressing the fusion proteins of the invention, which contain the coding sequences for the fusion proteins of the invention. The host cell is preferably a eukaryotic cell, such as but not limited to CHO, COS, 293, RSF, etc. In a preferred embodiment of the present invention, the cell is a CHO cell which can express the fusion protein of the present invention well and can obtain a fusion protein having a good binding activity and a good stability.

The present invention also provides a method for preparing the fusion protein of the present invention using recombinant DNA, comprising the steps of:

1) providing a nucleic acid sequence encoding a fusion protein (as set forth in SEQ ID NO: 3 sequence);

2) inserting the nucleic acid sequence of 1) into a proper expression vector to obtain a recombinant expression vector;

3) introducing the recombinant expression vector of 2) into a suitable host cell;

4) culturing the transformed host cell under conditions suitable for expression;

5) the supernatant was collected and the fusion protein product was purified.

Introduction of the coding sequence into a host cell can be accomplished by a variety of techniques known in the art, such as, but not limited to: calcium phosphate precipitation, protoplast fusion, lipofection, electroporation, microinjection, reverse transcription, phage transduction, and alkali metal ion.

For the culture and expression of host cells see Olander RM Dev Biol Stand 1996; 86: 338. the cells and debris in the suspension can be removed by centrifugation and the supernatant collected. Identification can be carried out by agarose gel electrophoresis techniques.

The fusion protein prepared as described above can be purified to substantially uniform properties, for example, as a single band on SDS-PAGE electrophoresis. For example, when the recombinant protein is expressed for secretion, the protein can be isolated using commercially available ultrafiltration membranes, such as Millipore, Pellicon, etc., and the expression supernatant is first concentrated. The concentrated solution can be further purified by gel chromatography or ion exchange chromatography. Such as anion exchange chromatography (DEAE etc.) or cation exchange chromatography. The gel matrix can be agarose, dextran, polyamide, etc. commonly used for protein purification. The Q-or SP-group is preferably an ion exchange group. Finally, the purified product can be further refined and purified by hydroxyapatite adsorption chromatography, metal chelate chromatography, hydrophobic interaction chromatography, reversed phase high performance liquid chromatography (RP-HPLC), and the like. All of the above purification steps can be combined in different ways to achieve a substantially uniform protein purity.

The expressed fusion protein can be purified using an affinity column containing an antibody, receptor or ligand specific for the fusion protein. The fusion polypeptide bound to the affinity column can be eluted by conventional methods, such as high salt buffer, pH change, etc., depending on the characteristics of the affinity column used. Optionally, the amino-terminus or the carboxy-terminus of the fusion protein may further comprise one or more polypeptide fragments as protein tags. Any suitable label may be used in the present invention. For example, the tag may be FLAG, HA, HA1, c-Myc, 6-His or 8-His, etc. These tags can be used to purify fusion proteins.

As a preferred mode of the present invention, the present inventors constructed the fusion protein gene, expressed it in CHO cells, and purified it to obtain a protein. Firstly, designing a fusion gene, constructing a recombinant plasmid containing a Glutamine Synthetase (GS) screening gene and a TNFR-IgG2/Fc gene by a method of primer synthesis and annealing splicing of a gene sequence, introducing the plasmid into CHO host cells by an electric shock transfection method, and selecting a cell strain which stably integrates a target gene and continuously expresses recombinant protein at a high level by screening the growth pressure of a medicament MSX in a culture medium. The cell strain is cultured in a serum-free commercial culture medium in a large scale, and the target protein with higher purity is obtained by protein A affinity chromatography and ion exchange chromatography. The inventor also verifies the physicochemical property verification and in vitro activity of the TNFR-IgG2/Fc fusion protein by using immunological principles and methods. The apparent molecular weight of the protein is verified by SDS-PAGE; structural characteristics of the TNFR-IgG2/Fc fusion protein of the target protein are proved by ELISA and Western-Blot, and the accuracy of the protein sequence is verified by N-terminal and C-terminal sequencing. The results of in vitro TNF alpha toxicity antagonism experiments show that the target protein and the TNF alpha molecules have strong binding capacity.

The invention also provides a composition comprising an effective amount (e.g., 0.000001-40 wt%, preferably 0.1-50 wt%, more preferably 5-40 wt%) of the fusion protein of the invention, and a pharmaceutically acceptable carrier.

Typically, the fusion proteins of the present invention can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5 to about 8, preferably about 6 to about 8.

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, a "pharmaceutically acceptable" component is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The compositions of the invention comprise a safe and effective amount of a fusion protein of the invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation is usually adapted to the administration mode, and the pharmaceutical composition of the present invention can be prepared in the form of injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the invention can also be prepared into a sustained release preparation.

The effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the fusion protein such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the fusion protein of the present invention is administered at a dose of about 0.00001mg to 50mg per kg of animal body weight (preferably 0.0001mg to 10mg per kg of animal body weight) per day. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

The fusion protein or the pharmaceutical composition containing the fusion protein can be used for binding TNF alpha, and thus can be used for preventing or treating diseases related to abnormal activation of TNF alpha. The diseases related to the abnormal activation of TNF alpha comprise but are not limited to: rheumatoid arthritis, ankylosing spondylitis, psoriasis, septicemia, asthma, stroke, diabetes, Crohn's disease, and a series of conditions caused by TNF-alpha molecules.

The fusion protein of the present invention or the pharmaceutical composition containing the fusion protein may also be administered in combination with other drugs. When used for preventing or treating diseases associated with abnormal activation of TNF alpha, the fusion protein may be administered systemically or locally, depending on factors such as the type, growth site, and degree of progression of the disease.

The main advantages of the invention are:

(1) the fusion protein can be specifically combined with human TNF alpha, effectively reduce the concentration of free TNF alpha in a human body and treat related diseases caused by the TNF alpha; the fusion protein not only retains the activity of binding TNF-alpha, but also has the characteristics of high TNF alpha affinity and low side effect (ADCC and CDC).

(2) The fusion protein of the invention has high stability and long in vivo half-life period, and overcomes the technical defect of poor stability of TNF alpha receptor preparations in the prior art.

The invention is further described below by way of examples, in which the experimental procedures, without specifying the particular conditions, are generally carried out according to conventional conditions, such as Sambrook et al, Molecular Cloning-A Laboratory Manual, Second Edition (Molecular Cloning-A Laboratory Manual, Second Edition), Cold spring harbor Laboratory Press, New York (1989) and the standard procedures described in Ausubel et al (1994) modern Molecular Biology techniques, Current Protocols in Molecular Biology, Current Protocols, Vol.

The following examples used carriers, species, reagents and sources:

pUC18-T, Extaq is a product of Takara corporation;

pfuultra hotspot DNA Polymerase is a product of Stratagene company;

the double expression vector pIRES is a product of Clontech company;

DNA ligase, restriction endonucleases MluI, XbaI, SalI and NotI are products of NEB company;

plasmid extraction, enzyme digestion product recovery and PCR product recovery were performed using the Purelink quick plasmid miniprep kit, Purelink quick gel extraction kit and Purelink PCR purification kit, respectively, from Invitrogen.

Example 1 acquisition of hTNFRII-Fc fusion Gene expression plasmid

Obtaining of hTNFRII-hIgG2/Fc Gene

The hTNFRII-hIgG2/Fc is a fusion protein, the gene sequence of which has 1389bp and codes 463 amino acid proteins. Wherein the sequence of the N-terminal hTNFRII extracellular region soluble fragment (mature protein) gene (705bp/235aa) is shown as SEQ ID NO: 1 (from Genebank NM 001066); c-terminal hIgG2-Fc gene (684bp/228aa), sequence SEQ ID NO: 2 (from Genebank AK 131045).

Oligonucleotide fragments of the synthetic genes were designed (table 1): the length of the fragment is about 70 bases generally, about 15 bases are complementary between two adjacent oligonucleotide fragments, and the annealing temperatures of complementary bases of the oligonucleotide fragments are ensured to be consistent as much as possible. The hTNFRII gene is assembled and synthesized by the method of overlap extension PCR.

TABLE 1 artificially synthesized hTNFRII-hIgG2/Fc oligonucleotide fragment sequence

Splicing and synthesizing sequence:

(1) fragments 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 were mixed, respectively, and overlap extension PCR was performed to 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, and 25-26.

And (3) PCR reaction system: dNTP 8 u l; 0.5. mu.l of Pfuultra enzyme; 10 μ l of 10 Xbuffer; DNA fragment 2X 20. mu.l; water 41.5. mu.l, total 100. mu.l. Circulation parameters: 94℃ × 5min → (94℃ × 30s → 60℃ × 30s → 2℃ × 45s) × 25 → 72℃ × 10 min.

(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 are respectively mixed and subjected to overlap extension PCR to obtain 1-4, 5-8, 9-12, 13-16, 17-20, 21-24 and 25-26.

Take 40. mu.l of each reaction (1) product, supplement 0.5. mu.l of Pfuultra enzyme, 20. mu.l of water, the cycle parameters were unchanged.

(3) 1-4, 5-8, 9-12, 13-16, 17-20 and 21-24 were mixed separately and subjected to overlap extension PCR to obtain 1-8, 9-16 and 17-24.

Take 40. mu.l of each reaction (2) product, supplement 0.5. mu.l of Pfuultra enzyme, 20. mu.l of water, the cycle parameters were unchanged.

(4) 1-8, 9-16, 17-24 and 25-26 were mixed and subjected to overlap extension PCR to obtain the full length of the gene.

Mu.l of each reaction product (3) was taken and supplemented with 0.5. mu.l of Pfuultra enzyme and 10. mu.l of water, with the cycle parameters unchanged.

(5) And amplifying the assembled gene by using the upstream and downstream primers to obtain a gene complete sequence.

And (3) PCR reaction system: dNTP 8 u l; 0.5. mu.l of Pfuultra enzyme; 10 μ l of 10 Xbuffer; 10. mu.l of DNA fragment; 2. mu.l of each primer; 67.5. mu.l of water, 100. mu.l total. Circulation parameters: 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. Acquisition of the Gene of Chinese hamster GS (glutathione synthase)

According to the GS gene sequence SEQ ID NO: 5(Genebank X03495), oligonucleotide fragments of the gene were synthesized, and the GS gene was synthesized by assembly by overlap extension PCR.

Construction of hTNFRII-Fc fusion Gene expression plasmid

(1) The SalI and NotI double-enzyme digestion GS gene and pIRES vector, and the reaction system comprises:

water bath at 37 ℃ for 2 h.

(2) Agarose gel electrophoresis, recovery of enzyme digestion products, the use of Purelink quick gel extraction kit, according to the kit instructions operation.

(3) Connecting a target gene with a vector, wherein the reaction system comprises:

water bath at 16 ℃ overnight.

(4) The ligation product was transformed into E.coli and cultured on LB plates. And selecting a plurality of clones for culture, extracting plasmid restriction enzyme identification on the next day, selecting positive clones for sequencing identification, and obtaining pIRES-GS recombinant plasmid with GS gene integrated in pIRES multiple cloning site B.

(5) The hIgG-Fc gene and the pIRES-GS vector are subjected to double enzyme digestion by MluI and XbaI, and the reaction system is the same as that in (1).

(6) Agarose gel electrophoresis, recovery of enzyme digestion products, the use of Purelink quick gel extraction kit, according to the kit instructions operation.

(7) The target gene and the vector are connected, and the reaction system is the same as (3).

(8) The ligation product was transformed into E.coli and cultured on LB plates. Selecting a plurality of clones for culture, extracting plasmid restriction enzyme identification (shown in figure 1) on the next day, selecting positive clones for sequencing identification, and obtaining the hTNFRII-hIgG/Fc fusion gene expression plasmid of which the hTNFRII-hIgG/Fc gene is integrated in the pIRES multiple cloning site A.

Example 2 expression and purification of hTNFRII-Fc fusion Gene

Transfection and selection of CHO cells

Cell lines and culture conditions: FreeStyle CHO-S cells (Invitrogen) cultured under FreeStyle medium (Invitrogen) with 10% dialyzed serum (Hyclone) and cultured in 10% CO₂And 37 ℃ incubator. G418, msx (sigma).

Lipofectamine manufactured by Invitrogen corporation was used for transfection^TM2000 Lipofectation kit, according to the instructions. Control CHO-S cells were transfected with empty pIRES vector. G418(200 mu M) and MSX (50 mu G/ml) are combined for pressure screening, the culture medium is replaced once in 3 days, the pressure is increased for 20 days, the culture medium is transferred to a 96-well plate for culture screening according to a limiting dilution method, and culture supernatant is taken for ELISA determination after 1 week of culture.

A mouse anti-human TNFR primary antibody (Sigma) is coated on an enzyme label plate (Corning) overnight at 4 ℃, 5% of milk is sealed, then cell culture supernatant is added, mouse anti-human IgG-Fc/HRP primary antibody (Sigma) is added after incubation, TMB reagent is developed after incubation, and OD value is measured at 450 nm. Fresh medium was used as a negative control. The ELISA standard curve prepared using TNFR-IgG2Fc standard is shown in FIG. 2. The results of the expression level of the antibody when each cell line was cultured in a 96-well plate are shown in Table 2.

TABLE 2 antibody expression level of each cell line cultured in 96-well plate

Sample (I)	Measured value (OD450)	Dilution factor	Titer (ng/ml)
				211p080723-2	2.06	100	13766
211p080723-3	2.181	100	20714

Sample (I)	Measured value (OD450)	Dilution factor	Titer (ng/ml)
				211p080723-4	2.212	100	24172
Negative control	0.046	100	Below the linear range

2. Single clone scale-up culture and fusion protein purification

Cell culture conditions: culturing in serum-free EX-CELL 302(SAFc) under 10% CO₂And 37 ℃ incubator.

Selecting a plurality of clones with the highest expression level to amplify to a 24-pore plate, detecting protein expression after culturing for 3 days, and selecting the clone with high expression to enter the next round of amplification; after multiple rounds of amplification, the 6 clones with the highest expression level were selected and expanded into T75 flasks, and the cell numbers were preserved. Inoculating the clone with the highest expression level into a 1L rotary bottle, culturing with EX-CELL 302 culture medium containing 5% calf serum, changing to serum-free culture medium when the CELL grows over the bottle wall, collecting liquid every other day, and continuously collecting liquid for 3-5 times.

The expressed protein was purified by affinity chromatography and ion exchange chromatography using the separation media rProtein A Sepharose4Fast flow (Amersham Biosciences) and Q Sepharose FF (Amersham Biosciences) using the specific adsorption of IgG by protein A, as shown in FIG. 3. The operation method refers to the product specification. After purification, the A260 and A280 values were determined by UV spectrophotometer. Protein quantification formula: protein content (mg/ml) ═ OD280 value × 1.45-OD260 value × 0.74.

Example 3 detection of the physicochemical Activity of hTNFRII-Fc fusion proteins

SDS-PAGE protein electrophoresis and Western blotting

20 μ l of the purified protein was subjected to SDS-PAGE, and the concentration of the gel was 10% as shown in FIG. 4. The detailed operation steps are described in the second edition of molecular cloning and protein electrophoresis experiment technology.

The samples were electrophoretically separated by SDS-PAGE and transferred to nitrocellulose membranes. The membrane was blocked with 5% milk solution containing 0.5% Tween, bound with antibody, primary antibody was mouse anti-human tnfr (sigma), secondary antibody was horseradish peroxidase-labeled goat anti-mouse igg (calbiochem), and after washing 3 times with PBS/0.5% Tween, TMB was developed, and the results are shown in fig. 5.

L929 cytotoxicity neutralization assay

Materials and reagents: l929(ATCC) in RPMI1640 (Sigma) containing 10% fetal bovine serum (Invitrogen) cultured in 5% CO₂And 37 ℃ incubator. TNF alpha (Sigma) was dissolved in RPMI1640 to 10000u/ml and dispensed at 0.1 ml/tube. Actinomycin D was dissolved to 1mg/ml with RPMI 1640. TNFR-Fc reference (standard): enbrel. The kit for counting the CCK-8 cells of the same kernel biochemistry.

(1) Collecting L929 cells in logarithmic growth phase, digesting, counting, adding into 96-well plate, 3 × 10⁵0.1 ml/well, incubated overnight.

(2) The following day 12ml of RPMI1640 was taken to dilute 0.12ml of actinomycin mother liquor to a final concentration of 10. mu.g/ml, 2ml was taken as a control (Medium A), and the remaining 10ml was added with TNF. alpha. mother liquor to a concentration of 10. mu.g/ml (Medium B).

(3) One aliquot of TNFR-Fc reference was taken, dissolved in 1ml of Medium B and diluted to a concentration of 256 ng/ml.

(4) According to the protein quantification of the samples to be tested, the samples were dissolved to 0.1mg/ml in sterile PBS and then diluted with medium B in a gradient in a 1.5ml sterile centrifuge tube.

(5) Dilutions of the reference or sample were serially diluted in multiple aliquots with medium in sterile centrifuge tubes, respectively. The volume of each dilution was 0.4 ml.

(6) Culture A was used as a negative control, and culture B was used as a positive control.

(7) Diluted samples, standards and negative and positive control solutions were added to the well plates inoculated with cells at 0.1 ml/well, with 3 duplicate wells for each measurement point. Continuously culturing in 5% CO2 and 37 deg.C incubator.

(8) After incubation for 5-10h, the medium was discarded, freshly prepared RPMI1640 containing 10% CCK-8 was added at 0.1 ml/well and incubation continued for 15 min.

(9) The reaction was stopped by adding 10. mu.l/well of 10% SDS and the value A450, referred to as A650, was measured with a microplate reader.

From the measurement results (as shown in fig. 6), it can be seen that in the TNF α in vitro toxicity binding experiment, the samples obtained from the multiple cell strains (211P80723-2, 211P80723-3, 211P80723-4) all have strong TNF α binding ability, and the TNF α antagonistic ability thereof is significantly higher than that of the same dose of Enbrel protein, which is 2.66, 1.26 and 1.26 times of the Enbrel activity, respectively, as shown in table 3.

TABLE 3 conclusion of TNF α in vitro toxicity binding experiments

Sample name	Enbrel	211P80723-2	211P080723-3	211P080723-4
					EC50 value (μ g/. mu.l)	0.0258	0.00971	0.0205	0.0205
Coefficient of correlation (R)2)	0.9973	0.9992	0.9963	0.9977
					EC50 working reference/EC 50 sample	/	2.657	1.259	1.259

Example 4 pharmacokinetic study of hTNFR II-Fc fusion protein in rats

504 rats are randomly divided into 6 groups, self-made samples are hTNFR II-IgG2/Fc fusion protein, control samples are commercial Wyeth company Enbrel, the hTNFR II-IgG2/Fc fusion protein is subcutaneously injected according to 0.5, 3 and 18mg/kg respectively, blood is collected at 14 times of snacks such as 1, 2, 4, 8, 12, 24, 28, 32, 36, 48, 72, 96, 120 and 144h after administration, 6 animals (3 male and 3 female) at each time point are centrifuged, serum is divided, the content of the hTNFR II in the serum is measured by a kit, the blood concentration at different times is calculated, pharmacokinetic and 3P87 software is used for carrying out c-t curve fitting on the data of the blood concentration (c) and the time (t) and calculating related pharmacokinetic parameters.

The rhTNFR blood concentration is measured by an enzyme labeling method at different time, the curve of the drug concentration-time data fitting treatment when the hTNFR II-IgG2/Fc drug is injected subcutaneously accords with the characteristic of single-chamber model non-vascular administration, and the main result parameters of three doses of 0.5, 3 and 18mg/kg are shown in Table 4. At the same dosage, hTNFR II-IgG2/Fc has a significantly higher metabolic cycle than Enbrel, and its half-life in vivo is 25.64% higher than that of Enbrel.

TABLE 4

t_1/2(ka)Represents: half-life of absorption;

t_1/2(ke)represents: elimination of half-life;

tpeak denotes: time to peak.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> Xinrun (Shanghai) biological pharmaceutical Co., Ltd

<120> TNFR-Fc fusion protein and application thereof

<130>085756

<160>33

<170>PatentIn version 3.3

<210>1

<211>235

<212>PRT

<213> Intelligent (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> Intelligent (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 (14)

1. A fusion protein, wherein the amino terminus of said fusion protein is a human tumor necrosis factor type II receptor soluble fragment; at the carboxy terminus is the Fc fragment of human immunoglobulin 2.

2. The fusion protein of claim 1, wherein the human tumor necrosis factor type II receptor soluble fragment has the amino acid sequence of SEQ ID NO: 1.

3. The fusion protein of claim 1, wherein the Fc fragment of human immunoglobulin 2 has the amino acid sequence of SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof.

4. A nucleic acid molecule encoding the fusion protein of any one of claims 1-3.

5. The nucleic acid molecule of claim 4, wherein said nucleic acid molecule has the sequence of SEQ ID NO: 3.

6. A vector comprising the nucleic acid molecule of claim 5.

7. The vector of claim 6, further comprising a gene encoding glutamine synthetase operably linked to said vector.

8. The vector of claim 7, wherein the glutamine synthetase has an amino acid sequence as set forth in SEQ ID NO: 4, respectively.

9. The vector of claim 7, wherein said vector is a pIRES bicistronic expression vector.

10. A genetically engineered cell characterized in that,

said cell comprising the vector of any one of claims 6-9; or

The cell genome having integrated therein the nucleic acid molecule of claim 4 or 5.

11. A method of producing the fusion protein of claim 1, comprising: culturing the host cell of claim 10 under conditions suitable for expression of the fusion protein, and expressing and isolating the fusion protein.

12. Use of a fusion protein according to any of claims 1 to 3 for the preparation of a composition specifically binding to tumor necrosis factor α.

13. The use according to claim 12, wherein said composition is used for the prevention or treatment of disorders associated with aberrant activation of tumor necrosis factor α.

14. A composition that specifically binds tumor necrosis factor α, said composition comprising:

(i) an effective amount of the fusion protein of any one of claims 1-3; and

(ii) a pharmaceutically acceptable carrier.

Images