CN115181185A

CN115181185A - CTGF-binding fusion protein and application thereof

Info

Publication number: CN115181185A
Application number: CN202110363638.1A
Authority: CN
Inventors: 张鹏; 马琳; 郭树华
Original assignee: Suzhou Pharmaceutical Technology Co ltd
Current assignee: Suzhou Pharmaceutical Technology Co ltd
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2022-10-14

Abstract

The invention provides a fusion protein, which is formed by connecting an anti-human CTGF antibody and a human type II TGF-beta receptor extracellular section through a joint, and can simultaneously combine CTGF and TGF-beta. The fusion protein can obtain the target protein with the purity of 100 percent after one-step purification, and simultaneously, the invention finds that the degree of pulmonary fibrosis of experimental animals can be greatly reduced even if very low dose of 1mg/kg is adopted for drug treatment in experiments.

Description

CTGF-binding fusion protein and application thereof

Technical Field

The invention belongs to the technical field of proteins, and particularly relates to a CTGF (total protein to fusion protein) combined fusion protein and application thereof.

Background

CTGF (connecting Tissue Growth Factor), also known as CCN2 or Connective Tissue Growth Factor, is a cytoplasmic protein of the CCN family of extracellular matrix-associated heparin-binding proteins. CTGF, as a cofactor involved in TGF-beta (Transforming Growth Factor-beta) signaling, can induce fibroblasts to become myofibroblasts, which deposit collagen, ultimately leading to organ scarring and dysfunction.

CTGF levels in tissues, blood or vitreous humor have been shown to be associated with fibrosis and its severity in a number of diseases. These two secreted factors of TGF- β and CTGF are widely recognized as universal mediators of fibrosis development. TGF-beta exerts functions of inhibiting cell proliferation and promoting extracellular matrix accumulation mainly through Smad and mitogen-activated protein kinase (MAPK). TGF-. Beta.1 can induce the synthesis of other cytokines such as platelet-derived growth factor, tumor necrosis factor, fibroblast growth factor, etc., and further promote the development of myocardial fibrosis by these factors [1]. When fibrosis of tubulointerstities occurs, extracellular matrix deposition increases, which is one of the causes of chronic renal failure. Renal interstitial fibrosis is a common pathway and pathological basis for the development of various kidney diseases to renal failure. Continued liver damage can lead to extensive fibrosis of the liver leading to cirrhosis. TGF-beta also plays a very important role in renal interstitial fibrosis and liver fibrosis. TGF- β is a potent inducer of CTGF, most models assume CTGF as a downstream mediator of TGF- β activity, while other studies support interdependencies between the respective profibrogenic activities of TGF- β and CTGF (interdependency), not just the sequential correlation [2]. Several animal data exist showing the synergistic effect of TGF- β and CTGF in the development and maintenance of a fibroblastic reaction.

Therapeutic applications using biological products such as anti-CTGF antibodies or interfering RNA for the field of fibrotic diseases such as pulmonary fibrosis, hepatic fibrosis, cancer, ophthalmological diseases, etc. are indicated in US20040248206A1, US20140343258A1, US201214367081, US201213385320, CN109402127A, CN109824777A, CN104011206A patents. The fibrotic disease is a disease of fibroblast mutation and tissue proliferation mediated by multiple cytokines, for example, TGF-beta, PDGF and IGF-1 are all fibrosis-causing growth factors in the process of pulmonary fibrosis, and are secreted and increased in early stage, and simultaneously, the process of pulmonary fibrosis is started, and irreversible lung injury is formed.

There is still a need in the art to provide new molecular entities to further optimize the treatment of diseases.

[1] (Dingwenjin et al, "role of TGF-. Beta.1 in myocardial fibrosis due to different causes" [ science and technology bulletin 2 (2016): 221-225 ])

[2](José G.Abreu,et al."Connective-tissue growth factor(CTGF)modulates cell signalling by BMP and TGF-β."Nature Cell Biology 4.8(2002):599-604.；Oganesian,A.,Y.Zhu,and L.J.Sandell."Type IIA procollagen amino propeptide is localized in human embryonic tissues."Journal of Histochemistry&Cytochemistry 45.11(1997):1469-1480.

Disclosure of Invention

In order to solve the above problems, the present invention provides a novel bifunctional fusion protein that can simultaneously bind CTGF and TGF-. Beta.s. Surprisingly, the fusion protein of the invention can obtain the target protein with extremely high purity (100%) after one-step purification, and the degree of pulmonary fibrosis of experimental animals can be greatly reduced even if the drug treatment is carried out by using very low dose (1 mg/kg) in experiments.

Interpretation of terms

In the present invention, unless defined otherwise, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

Exemplary techniques for use in conjunction with recombinant DNA, oligonucleotide synthesis, tissue culture and transformation (e.g., electroporation, lipofection), enzymatic reactions, and purification techniques are known in the art. Many such techniques and procedures are described, for example, in Sambrook et al, molecular Cloning: a Laboratory Manual (2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y. (1989)), and many others. In addition, exemplary techniques for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients are also known in the art.

In this application, the use of "or" means "and/or" unless stated otherwise. In the case of multiple dependent claims, the use of "or" in the alternative merely refers to more than one of the foregoing independent or dependent claims.

As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood as including the value of any integer within the range, and where appropriate including fractions thereof (such as tenths and hundredths of integers), unless otherwise indicated.

Units, prefixes, and symbols are denoted in their international system of units (SI) recognized form. Numerical ranges include the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure which can be had by reference to the specification as a whole.

Unless otherwise indicated, as used in accordance with this disclosure, the following terms are to be understood to have the following meanings:

"antibody" refers to a molecule comprising at least the complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain and at least the CDR1, CDR2 and CDR3 of a light chain, and further comprising the framework regions FR1, FR2, FR3 and FR4, wherein the molecule is capable of binding to an antigen. The term antibody includes, but is not limited to, fragments capable of binding antigen, such as Fv, single chain Fv (scFv), fab ', and (Fab') 2. The term antibody also includes, but is not limited to, chimeric antibodies, humanized antibodies, and antibodies of various species, such as mouse, human, cynomolgus monkey, alpaca, and the like.

In some embodiments, the antibody comprises a heavy chain variable region and a light chain variable region. In some embodiments, the antibody comprises at least one heavy chain comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, the antibody comprises two heavy chains and two light chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region. Any other antibody comprising a single polypeptide chain comprising, for example, all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have one heavy chain and one light chain. In some such embodiments, the heavy chain is an antibody region comprising three heavy chain CDRs and the light chain is an antibody region comprising three light chain CDRs.

"complementarity determining regions" or "CDRs" are regions in an antibody variable domain that are mutated in sequence and form structurally defined loops ("hypervariable loops") and/or regions containing antigen-contacting residues ("antigen-contacting points"). The CDRs are primarily responsible for binding to an epitope of the antigen. The CDRs of the heavy and light chains are commonly referred to as CDR1, CDR2 and CDR3, numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2 and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2 and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of the CDRs may be determined using any one or combination of a number of well-known antibody CDR assignment systems, including, for example: chothia (Chothia et al (1989) Nature 342-883, al-Lazikani et al, "Standard constraints for the structural of the pathological structures of immunoglobulins", journal of Molecular Biology,273,927-948 (1997)), kabat (Kabat et al, sequences of Proteins of Immunological Interest, 4 th edition, U.S. department of Health and Human Services, national Institutes of Health (1987)), abM (version of balance), unity (University Collection), international Munongeneric standards database (IMG), and the use of the structural of the Crystal (CDR) in large numbers, as well as the clustering of the structural of the CDRs (neighboring propagation definitions). The CDRs in the present invention are divided according to the Kabat et al protocol.

In some embodiments, the amino acid alterations described herein comprise substitutions, insertions, or deletions of amino acids. Preferably, the amino acid changes described herein are amino acid substitutions, preferably conservative substitutions.

In a preferred embodiment, the amino acid changes described herein occur in regions outside the CDRs (e.g., in the FRs). More preferably, the amino acid changes of the invention occur in regions outside the heavy chain variable region and/or outside the light chain variable region.

In some embodiments, the substitution is a conservative substitution. Conservative substitutions are those where one amino acid is substituted with another within the same class, for example, where one acidic amino acid is substituted with another acidic amino acid, one basic amino acid is substituted with another basic amino acid, or one neutral amino acid is substituted with another neutral amino acid. Exemplary permutations are shown in the following table:

"Fc" or "Fc fragment" refers to the "fragment crystallizable" region of an immunoglobulin heavy chain. In general, an Fc domain is capable of interacting with another Fc domain to form a dimeric complex. The Fc domain may bind to a cell surface receptor (Fc receptor) and/or a protein of the complement system, or may be modified to impair or enhance such binding activity. The Fc domain may be derived from IgG, igA, igD, igM, or IgE antibodies and produce immune functions such as opsonization, cell lysis, mast cell degranulation, among other Fc receptor dependent processes. The original immunoglobulin source of the native Fc is preferably an immunoglobulin of human origin, preferably IgG1, igG2 and IgG4.

In certain embodiments, an "Fc mutant" refers to a molecule or sequence that is modified by a native Fc and can still bind an Fc receptor. "Fc mutants" include molecules or sequences humanized from non-human native Fc "Fc domains" include molecules or sequences of native Fc and Fc variants as described above, which comprise molecules in monomeric or multimeric form, which can be obtained by whole antibody dissociation, or by gene recombinant expression or by other means.

"host cell" refers to a cell that may be or has been the recipient of a vector or isolated polynucleotide. The host cell may be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; a plant cell; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, and 293 and CHO cells and derivatives thereof, such as 293-6E and DG44 cells, respectively.

"coronavirus" is named by its surface crown-shaped spike. There are four major subgroups of coronaviruses, called α, β, γ, and δ. The seven coronaviruses that infect humans are: 229E (alphacoronavirus), NL63 (alphacoronavirus), OC43 (beta coronavirus), HKU1 (beta coronavirus), middle east respiratory syndrome coronavirus (MERS-CoV), SARS coronavirus (beta coronavirus causing severe acute respiratory syndrome or SARS, also known as SARS-CoV), SARS-CoV-2 (a novel coronavirus causing coronavirus disease in 2019, or new coronaviridae). Humans around the world are often infected with human coronavirus 229E, NL63, OC43, and HKU1. Sometimes animal-infected coronaviruses evolve to make human diseases new to human coronaviruses. The last three examples are 2019-nCoV, SARS-CoV and MERS-CoV. Severe pneumonia and severe acute pneumonia of coronavirus can cause cytokine storm, severe hyperimmune inflammatory reaction and tissue destruction occur in lung tissue, and secondary pulmonary fibrosis is possible.

"fibrotic diseases" are connective tissue diseases such as systemic sclerosis, in which excessive activation of connective tissue cells leads to tissue hardening and scarring within the affected organ. In principle, these diseases can affect any organ system and often lead to a disruption of organ function. Fibrosis can occur in a variety of organs or tissues, such as the liver, kidneys, lungs, heart, pancreas, eye, skin, joints, etc., and is the basis for the pathogenesis of many diseases. Fibrotic diseases include liver fibrosis, cirrhosis, nonalcoholic steatohepatitis, hepatolenticular degeneration, idiopathic pulmonary fibrosis, renal fibrosis, myocardial fibrosis, scleroderma, toxic oil syndrome, cutaneous fibroma, keloids, hypertrophic scars, bone fibrosis, intestinal fibrosis, cystic Fibrosis (CF), duchenne Muscular Dystrophy (DMD), pseudohypertrophic muscular dystrophy, becker guanidine dystrophy, emery-Drcifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, myotonic dystrophy, and the like.

"cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

"tumor" refers to all neoplastic (neoplastic) cell growth and proliferation, whether malignant or benign, and all pre-cancerous (pre-cancerous) and cancerous cells and tissues. The terms "cancer," "cancerous," "cell proliferative disorder," "proliferative disorder," and "tumor" are not mutually exclusive when referred to herein.

"cancer" is customarily used to refer broadly to all malignant tumors. Examples of cancers include, but are not limited to: carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific non-limiting examples of such cancers include squamous cell cancer, small-cell lung cancer, pituitary cancer, esophageal cancer, astrocytoma, soft tissue sarcoma, non-small cell lung cancer (including squamous cell non-small cell lung cancer), adenocarcinoma of the lung, squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, uterine cancer, salivary gland carcinoma, kidney cancer, renal cell carcinoma, prostate cancer, vulval cancer, thyroid cancer, brain cancer, endometrial cancer, testicular cancer, biliary tract cancer, gallbladder cancer, gastric cancer, melanoma, and various types of head and neck cancer (including head and neck squamous cell carcinoma).

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathological condition or disorder. In certain embodiments, the term "treatment" encompasses any administration or use of a disease therapeutic in a mammal (including a human), and includes inhibiting or slowing the disease or progression of the disease; partial or complete response to disease, e.g., by causing regression, or recovery or repair of lost, lost or defective function; a stimulus-ineffective process; or to smooth the disease to a reduced severity. The term "treating" also includes reducing the severity of any phenotypic characteristic and/or reducing the incidence, extent or likelihood of that characteristic. Those in need of treatment include those already with the disorder as well as those predisposed to having the disorder, or those in which the disorder is to be prevented.

In one aspect, the invention provides a fusion protein.

The fusion protein is formed by connecting an anti-human CTGF antibody and the extracellular section of a human type II TGF-beta receptor through a joint.

The sequence of the CTGF is SEQ ID NO.1.

The sequence of the human type II TGF-beta receptor is SEQ ID NO.2.

The human type II TGF-beta receptor extracellular section of the fusion protein is selected from a sequence SEQ ID NO.3 or SEQ ID NO.4.

The CDR of the light chain variable region of the antihuman CTGF antibody is selected from SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10.

Preferably, the light chain variable region of said anti-human CTGF antibody is selected from SEQ ID No.11 or SEQ ID No.12, or an amino acid sequence having at least 90% identity to SEQ ID No.11 or SEQ ID No. 12. Wherein the CDR sequences of the light chain variable region of SEQ ID NO.11 are SEQ ID NO.5 (LCDR 1), SEQ ID NO.6 (LCDR 2) and SEQ ID NO.7 (LCDR 3), respectively; the CDR sequences of the light chain variable region of SEQ ID NO.12 are SEQ ID NO.8 (LCDR 1), SEQ ID NO.9 (LCDR 2) and SEQ ID NO.10 (LCDR 3), respectively.

Preferably, the light chain constant region of said anti-human CTGF antibody is SEQ ID No.21 or an amino acid sequence having at least 90% identity to SEQ ID No. 21.

Or the CDR of the heavy chain variable region of the anti-human CTGF antibody is selected from SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, wherein the amino acid C in SEQ ID NO.15 can be replaced, and the preferable amino acid C can be replaced by the amino acids S, Y and F.

Preferably, the heavy chain variable region of said anti-human CTGF antibody is selected from the group consisting of SEQ ID NO.19 or SEQ ID NO.20, or an amino acid sequence having at least 90% identity to SEQ ID NO.19 or SEQ ID NO. 20. Wherein, the CDR sequences of the heavy chain variable region of SEQ ID NO.19 are respectively SEQ ID NO.13 (HCDR 1), SEQ ID NO.14 (HCDR 2) and SEQ ID NO.15 (HCDR 3); the CDR sequences of the heavy chain variable region of SEQ ID NO.20 are SEQ ID NO.16 (HCDR 1), SEQ ID NO.17 (HCDR 2) and SEQ ID NO.18 (HCDR 3), respectively.

The heavy chain constant region of the antihuman CTGF antibody in the fusion protein is selected from the constant regions of human IgG1, igG2 and IgG4.

The joint of the fusion protein is selected from (G) _x S _y ) _n A linker wherein x is 0, 1, 2, 3, 4, 5, y is 0, 1, 2, 3, 4, 5, n is 0, 1, 2, 3, 4, 5.

The human TGF-beta receptor extracellular segment is connected with the N end or the C end of an anti-human CTGF antibody heavy chain to form a fusion protein, or the human TGF-beta receptor extracellular segment is connected with the N end or the C end of an anti-human CTGF antibody light chain to form a fusion protein; preferably, the human TGF-beta receptor extracellular segment forms a fusion protein after being connected with the C end of the heavy chain of the anti-human CTGF antibody.

The fusion protein can completely or partially block the combination of CTGF and TGF-beta and the receptor thereof.

In another aspect, the invention provides a nucleic acid molecule.

The nucleic acid molecule encodes a fusion protein as described above.

In yet another aspect, the present invention provides an expression vector.

The expression vector is used for transfecting a mammalian cell, and the expression vector comprises the nucleic acid molecule.

In yet another aspect, the invention provides a host cell.

The host cell comprises the aforementioned expression vector.

In yet another aspect, the present invention provides a pharmaceutical composition.

The pharmaceutical composition comprises the aforementioned fusion protein and/or nucleic acid molecule and/or expression vector and/or host cell.

The pharmaceutical composition also comprises other pharmaceutically acceptable carriers.

In a further aspect, the invention provides the use of the aforementioned fusion protein and/or nucleic acid molecule and/or expression vector and/or host cell and/or pharmaceutical composition for the prevention or treatment of a tissue proliferative disease, cancer or fibrotic disease.

The therapeutic use of said tissue proliferative disease, cancer or fibrotic disease is selected from the group consisting of:

<xnotran> , , , , , , , , , , , , , , , , , , , , goodpasture ( - ), , , , , ( ), , , , , , whipple ( ), , (Crohn) , , , , langerhans , , , , , , , , , , , , , , , , , , , , , , , , , , becker , emery-Drcifuss , </xnotran> Facioscapulohumeral muscular dystrophy, myotonic dystrophy, glaucoma filtration surgery fibrosis, macular degeneration, diabetic retinopathy, choroidal neovascularization, proliferative vitreoretinopathy and wound healing.

The disease is pulmonary fibrosis caused by coronavirus or other respiratory virus infection.

The fusion protein, the nucleic acid molecule, the expression vector or the host cell and the second pharmacologically active compound are used separately, sequentially or simultaneously.

In yet another aspect, the invention provides a method of preparing a fusion protein.

The fusion protein is the fusion protein, and the preparation method comprises the following steps:

(1) Splicing the light chain and the heavy chain of the antibody respectively;

(2) Cloning the light chain amino acid sequence and the heavy chain amino acid sequence of the antibody to a vector; selecting clone for sequencing, selecting correctly sequenced thallus for preserving the strain and performing enlarged culture of the thallus, wherein the enlarged thallus is used for extracting plasmid;

(3) Separating and purifying after culturing the plasmid transformed cells.

The invention has the advantages that:

1. the fusion protein of the invention can obtain the target protein with extremely high purity (100%) after one-step purification.

2. By adopting the fusion protein provided by the invention, 1mg/kg of the fusion protein can greatly reduce the degree of pulmonary fibrosis of experimental animals when used for drug treatment.

Drawings

FIG. 1 shows the SEC-HPLC detection results of the fusion protein in example 2.

FIG. 2 is a map of affinity fit of the fusion protein to human CTGF in example 3.

FIG. 3 is a graph of the affinity fit of the fusion proteins to human TGF-. Beta.s in example 3.

FIG. 4 is a pathological section of lung tissue of a control group mouse in an animal drug effect experiment of fusion protein.

FIG. 5 shows lung tissue pathological sections of untreated mice in the drug effect experiment of fusion protein animals.

FIG. 6 is a pathological section of lung tissue of mice treated with low dose in the drug effect experiment of fusion protein animals.

FIG. 7 is a pathological section of lung tissue of high-dose treatment group mice in a fusion protein animal drug effect experiment.

Detailed Description

The present invention will be described in further detail with reference to specific examples, which are not intended to limit the present invention, but to illustrate the present invention. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.

EXAMPLE 1 molecular construction of fusion proteins

The partial experiment is completed by Suzhou Jinwei Zhi Biotechnology Co., ltd, and comprises the following steps:

(1) Splicing SEQ ID NO.11 and SEQ ID NO.21 to form a light chain of the antibody;

(2) Splicing SEQ ID No.22 and SEQ ID No.23 before connecting to SEQ ID No.4 through SEQ ID No.24 to form the heavy chain of the antibody;

(3) The light and heavy chain amino acid sequences of the above antibodies were codon-optimized according to human host cells and the genes were synthesized conventionally, and 5 '(EcoRI) and 5' UTR (SEQ ID NO. 25) and 3'UTR (TGATGA) and 3' (HindIII) were added to clone the genes into vector pTT5 (Ampicillin) through 5'EcoRI and 3' HindIII. Selecting clone for sequencing, selecting correctly sequenced thallus for conservation and enlarged culture, and using the enlarged thallus for extraction of plasmid.

EXAMPLE 2 expression and purification of fusion proteins

Cell transfection and protein isolation and purification of the extracted plasmid were performed as follows:

(1) Measurement of cell Density and viabilityShould be more than 95%, the cell density was adjusted to 3X 10 with a pre-warmed 293 cell culture medium (purchased from Ovapmy Biotech Co., ltd., shanghai, under the designation P82019) ⁶ cells/mL, gently shaken and aliquoted (transfection system 90%), the volume of cells in the flask does not exceed 1/3 of the flask specification, and placed into a shaker for use.

(2) The volume of the transfection buffer opti-MEM (purchased from Saimer Feishell technology (China) Co., ltd., product number 11058021) was calculated based on the volume of the transfected cells, and was 1/10 of that of the transfection system; calculating the amount of PEI (purchased from Saimer Feishale science, inc., china, cat. BMS 1003) as a transfection reagent at a ratio of 3. Mu.L/mL; the total amount of transfected DNA was calculated in a ratio of 1. Mu.g/mL of transfected cells.

The specific transfection procedure was as follows:

(1) And adding 1 piece of 50mL centrifuge tube into opti-MEM with the concentration of 10% in the transfection system, adding the plasmid, uniformly mixing, filtering, standing for 5min, adding PEI into the DNA suspension, gently mixing (gently inverting and uniformly mixing for 2-3 times), and standing for 15-20min. Then, the compound is gently added into the subpackaged cells, and the shake flask is gently shaken while the compound is added; the transfected cells were cultured in a shaker at 37 ℃.

(2) Feeding on day 1: after 20h, cells were transfected at 1.

(3) Determining the expression amount of the transfected cells on day 4: 200 mu L of sample to be tested is taken, a label-free analyzer is used for detecting the protein expression quantity, 293 culture medium is used as a negative control, and a solution with known protein concentration is used as a positive control during the analysis of the instrument.

(4) Collecting samples on 5-6 days: detecting the cell viability (65-75% or higher is preferable), centrifuging the sample at 3000-5000 Xg for 20-30 min, filtering the supernatant with 0.22 μm filter, and separating, purifying and detecting the obtained supernatant sample, wherein FIG. 1 is SEC-HPLC detection result of fusion protein.

Wherein, purification is carried out according to the product specification, which is briefly as follows:

1) Pre-balancing: by ddH ₂ O-Wash HiTrap Protein L (general electric medical systems, inc.; product number 29048665) column5CV, punching out protective solution; and (5) balancing the chromatographic column by using a balance liquid, wherein the ultraviolet detection base line is kept stable.

2) Loading: the sample is loaded and the retention time is 4min. When the ultraviolet detection value rises, the flow-through starts to be collected.

3) And (3) post-balancing: and (5) after the sample loading is finished, balancing 5-10CV by using a balance liquid until the ultraviolet detection base line is stable, and stopping collecting and penetrating.

4) And (3) elution: eluting with 3-5CV of eluent and neutralizing solution, respectively, and collecting the eluate.

5) Column washing: washing the column with regeneration buffer for 15min, and then washing with ddH ₂ O flush the regeneration buffer out until the pH is neutral.

The purity measurement was carried out as follows:

an Agilent liquid phase detection system (Agilent technologies, inc., model 1200) is combined with an SEC-300 type analytical column, a sample is centrifuged, and then a supernatant is taken and filtered by a 0.22 mu m filter, 20 mu L of the supernatant is loaded, and the flow rate is 0.26mL/min. At 10.632min, the peak area of the main peak detected by the instrument was 2392.859mAU, and the peak area ratio was 100%.

Example 3 binding assays of fusion proteins with human CTGF and human TGF-beta proteins

By using Gator ^TM The above-expressed fusion proteins were subjected to affinity detection with human CTGF protein (purchased from ACROBIOSystems, cat # CTF-H52H 5) and human TGF-. Beta.protein (purchased from ACROBIOSystems, cat # TG 1-H4212), respectively, using a non-labeled bioanalyzer. And (3) capturing the antibody sample by using a Protein A biosensor, and then performing dynamic detection of combination and dissociation on the captured antibody sample and human CTGF Protein and human TGF-beta Protein respectively. Kinetics were performed using 1:1 the fitting analysis is performed in conjunction with the model. The brief steps are shown in the following table:

step (ii) of	Time
		Protein loading	200s
Bonding with	180s
		Dissociation	300s
Regeneration	30s

Using Gator ^TM The affinity data measured by the instrument are shown in the following table:

capturing a sample	Binding sample	Response value	Affinity (M)	Dissociation constant (1/s)	Binding constant (1/Ms)
						Fusion proteins	Human CTGF	0.0665	5.40E-08	1.23E-02	2.28E+05
Fusion proteins	Human TGF-beta	0.142	2.84E-09	1.82E-03	6.40E+05

The maps corresponding to the affinity assays for human CTGF and human TGF- β of the fusion proteins are shown in FIGS. 2 and 3.

The detection of the affinity of the fusion protein with human CTGF and human TGF-beta shows that the fusion protein provided by the invention can be normally combined with human CTGF and human TGF-beta, and on the basis, the fusion protein can play a role in blocking signal pathways of human CTGF and human TGF-beta and inhibit or slow down the process of tissue fibrosis.

Example 4 animal drug efficacy test of fusion protein

4.1 Main test materials

BALB/c mice (purchased from Experimental animals technology, inc., viton, beijing, strain code 211).

Bleomycin (available from Shanghai, 25035kang Biotech, inc., cat No. MZ 3504).

Chloral hydrate (available from national pharmaceutical group chemical agents, ltd., cat # 30037516).

4.2 model building

BALB/c mice were anesthetized by intraperitoneal injection using 4% chloral hydrate in a volume of 200. Mu.L, and then the mice were administered by nasal drip using a pipette with 30. Mu.L of a bleomycin solution with a concentration of 3.33mg/mL or 30. Mu.L of a PBS solution, and the converted mouse model administration dose was 5mg/kg. During the administration process, a mouse under general anesthesia is fixed by the left hand, the body of the mouse is kept in a vertical state of head up and tail down, a micropipettor is held by the right hand, 30 mu L of the bleomycin solution is gently dripped at the nostril of the mouse, and if the mouse breathes normally and gradually inhales liquid, the nasal dripping operation can be continued until all the medicines enter the nasal cavity. On day 7 of molding, therapeutic administration of the tail vein of the mouse was started, and the protein for therapeutic administration was the purified protein of example 2, administered once a week for three times. Dosing and treatment groups are shown in the following table:

group of	Molding reagent	Therapeutic administration
			Control group	30μL PBS	Without any treatment
Untreated group	30 mu.L of bleomycin solution	PBS
			Low dose treatment group	30 μ L of bleomycin solution	1mg/kg, fusion protein
High dose treatment group	30 μ L of bleomycin solution	10mg/kg, fusion protein

After treatment and administration are finished, the lung tissues of the mice are taken and fixed by 4% paraformaldehyde, and after the fixation state is good, trimming, dehydrating, embedding, slicing, dyeing, mounting and finally microscopic examination are carried out on qualified sample wafers. And (4) viewing the section under a microscope or viewing a digital section, and observing the condition of the tissue section in detail under different multiples.

4.3 pathological detection and analysis

Pathological section staining showed that all mice dosed with bleomycin developed varying degrees of pulmonary fibrosis, but mice modelled with PBS had normal lungs.

The lungs of mice that were modeled with bleomycin alone but not treated showed typical and severe pulmonary fibrosis, which was characterized by massive collagen fibroplasia in the lung tissue sections, thickening of alveolar walls, indistinguishable alveolar spaces and coverage of fibrotic mass.

Meanwhile, section staining showed that the fusion protein treatment administration group showed a reduction in the degree of pulmonary fibrosis, and a low dose fusion protein treatment administration group of 1mg/kg also obtained a significant improvement in pulmonary fibrosis. The fusion protein in the invention can be used for effectively treating fibrotic diseases through in vivo drug effect tests of animals.

Examples 5-8 molecular construction of fusion proteins

A fusion protein was prepared by the method of reference example 1-2, except that the light chain variable region, the heavy chain variable region and the extracellular domain of the human type II TGF-beta receptor were different in sequence, and the specific sequence information was as follows:

	light chain variable region	Heavy chain variable region	Extracellular domain of TGF-beta receptor of human type II
				Example 5	SEQ ID NO:11	SEQ ID NO:19	SEQ ID NO:3
Example 6	SEQ ID NO:12	SEQ ID NO:19	SEQ ID NO:4
				Example 7	SEQ ID NO:11	SEQ ID NO:20	SEQ ID NO:3
Example 8	SEQ ID NO:12	SEQ ID NO:20	SEQ ID NO:3

The fusion proteins of examples 5 to 8 were compared with the experimental methods of examples 3 to 4, and the results showed that the properties of the fusion proteins prepared by the methods of examples 1 to 2 were substantially identical.

Comparative example

The fusion proteins were prepared by referring to the methods of examples 1-2, except that the linker sequences were SEQ ID NO.26, SEQ ID NO.27, and SEQ ID NO.28, respectively, to obtain fusion protein 1, fusion protein 2, and fusion protein 3.

The obtained purity of the fusion protein 1 was 95.8%.

Fusion protein 2 was obtained with a purity of 96.4%.

Fusion protein 3 was obtained with a purity of 97.7%.

After further purification of the fusion proteins 1, 2 and 3, the experiment was carried out by referring to the method of example 4, and the following results were obtained:

sequence listing

<110> Suzhou pulekang pharmaceutical science and technology Co., ltd

<120> CTGF-binding fusion protein and application thereof

<160> 28

<170> SIPOSequenceListing 1.0

<210> 1

<211> 322

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asn Cys Ser Gly Pro Cys Arg Cys Pro Asp Glu Pro Ala Pro Arg Cys

1 5 10 15

Pro Ala Gly Val Ser Leu Val Leu Asp Gly Cys Gly Cys Cys Arg Val

20 25 30

Cys Ala Lys Gln Leu Gly Glu Leu Cys Thr Glu Arg Asp Pro Cys Asp

35 40 45

Pro His Lys Gly Leu Phe Cys His Phe Gly Ser Pro Ala Asn Arg Lys

50 55 60

Ile Gly Val Cys Thr Ala Lys Asp Gly Ala Pro Cys Ile Phe Gly Gly

65 70 75 80

Thr Val Tyr Arg Ser Gly Glu Ser Phe Gln Ser Ser Cys Lys Tyr Gln

85 90 95

Cys Thr Cys Leu Asp Gly Ala Val Gly Cys Met Pro Leu Cys Ser Met

100 105 110

Asp Val Arg Leu Pro Ser Pro Asp Cys Pro Phe Pro Arg Arg Val Lys

115 120 125

Leu Pro Gly Lys Cys Cys Glu Glu Trp Val Cys Asp Glu Pro Lys Asp

130 135 140

Gln Thr Val Val Gly Pro Ala Leu Ala Ala Tyr Arg Leu Glu Asp Thr

145 150 155 160

Phe Gly Pro Asp Pro Thr Met Ile Arg Ala Asn Cys Leu Val Gln Thr

165 170 175

Thr Glu Trp Ser Ala Cys Ser Lys Thr Cys Gly Met Gly Ile Ser Thr

180 185 190

Arg Val Thr Asn Asp Asn Ala Ser Cys Arg Leu Glu Lys Gln Ser Arg

195 200 205

Leu Cys Met Val Arg Pro Cys Glu Ala Asp Leu Glu Glu Asn Ile Lys

210 215 220

Lys Gly Lys Lys Cys Ile Arg Thr Pro Lys Ile Ser Lys Pro Ile Lys

225 230 235 240

Phe Glu Leu Ser Gly Cys Thr Ser Met Lys Thr Tyr Arg Ala Lys Phe

245 250 255

Cys Gly Val Cys Thr Asp Gly Arg Cys Cys Thr Pro His Arg Thr Thr

260 265 270

Thr Leu Pro Val Glu Phe Lys Cys Pro Asp Gly Glu Val Met Lys Lys

275 280 285

Asn Met Met Phe Ile Lys Thr Cys Ala Cys His Tyr Asn Cys Pro Gly

290 295 300

Asp Asn Asp Ile Phe Glu Ser Leu Tyr Tyr Arg Lys Met Tyr Gly Asp

305 310 315 320

Met Ala

<210> 2

<211> 112

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ala Leu Asp Thr Asn Tyr Cys Phe Ser Ser Thr Glu Lys Asn Cys Cys

1 5 10 15

Val Arg Gln Leu Tyr Ile Asp Phe Arg Lys Asp Leu Gly Trp Lys Trp

20 25 30

Ile His Glu Pro Lys Gly Tyr His Ala Asn Phe Cys Leu Gly Pro Cys

35 40 45

Pro Tyr Ile Trp Ser Leu Asp Thr Gln Tyr Ser Lys Val Leu Ala Leu

50 55 60

Tyr Asn Gln His Asn Pro Gly Ala Ser Ala Ala Pro Cys Cys Val Pro

65 70 75 80

Gln Ala Leu Glu Pro Leu Pro Ile Val Tyr Tyr Val Gly Arg Lys Pro

85 90 95

Lys Val Glu Gln Leu Ser Asn Met Ile Val Arg Ser Cys Lys Cys Ser

100 105 110

<210> 3

<211> 136

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Ile Pro Pro His Val Gln Lys Ser Val Asn Asn Asp Met Ile Val Thr

1 5 10 15

Asp Asn Asn Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp

20 25 30

Val Arg Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys

35 40 45

Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val

50 55 60

Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Asp

65 70 75 80

Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro

85 90 95

Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met

100 105 110

Cys Ser Cys Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu

115 120 125

Glu Tyr Asn Thr Ser Asn Pro Asp

130 135

<210> 4

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Gly Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys Asp Val Arg Phe

1 5 10 15

Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn Cys Ser Ile Thr

20 25 30

Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala Val Trp Arg Lys

35 40 45

Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His Glu Pro Lys Leu

50 55 60

Pro Tyr His Asp Phe Ile Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile

65 70 75 80

Met Lys Glu Lys Lys Lys Pro Gly Glu Thr Phe Phe Met Cys Ser Cys

85 90 95

Ser Ser Asp Glu Cys Asn Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn

100 105 110

Thr Ser Asn Pro Asp

115

<210> 5

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Arg Ala Ser Gln Gly Ile Ser Ser Trp Leu Ala

1 5 10

<210> 6

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 7

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gln Gln Tyr Asn Ser Tyr Pro Pro Thr

1 5

<210> 8

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Arg Ala Ser Gln Ser Val Ser Ser Ser Tyr Leu Ala

1 5 10

<210> 9

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Gly Ala Ser Arg Arg Ala Thr

1 5

<210> 10

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Gln Gln Tyr Val Ser Thr Pro Trp Thr

1 5

<210> 11

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 11

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 12

<211> 108

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 12

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser

20 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

35 40 45

Ile Tyr Gly Ala Ser Arg Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser

50 55 60

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

65 70 75 80

Pro Glu Asp Phe Val Val Tyr Phe Cys Gln Gln Tyr Val Ser Thr Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys

100 105

<210> 13

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 13

Ser Tyr Gly Met His

1 5

<210> 14

<211> 16

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 14

Gly Ile Gly Thr Gly Gly Gly Thr Tyr Ser Thr Asp Ser Val Lys Gly

1 5 10 15

<210> 15

<211> 12

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 15

Gly Asp Tyr Tyr Gly Ser Gly Ser Phe Phe Asp Cys

1 5 10

<210> 16

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 16

Gly Tyr Tyr Met Tyr

1 5

<210> 17

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 17

Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 18

<211> 10

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 18

Gly Ser Lys Trp Asn Tyr Pro Phe Asp Tyr

1 5 10

<210> 19

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 19

Glu Gly Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Gly Thr Gly Gly Gly Thr Tyr Ser Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Asp Tyr Tyr Gly Ser Gly Ser Phe Phe Asp Cys Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 20

<211> 119

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 20

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Leu Tyr Tyr Cys

85 90 95

Ala Arg Gly Ser Lys Trp Asn Tyr Pro Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210> 21

<211> 107

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 22

<211> 120

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Gly Ile Gly Thr Gly Gly Gly Thr Tyr Ser Thr Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Gly Asp Tyr Tyr Gly Ser Gly Ser Phe Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 23

<211> 329

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 24

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 25

<211> 66

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

gccaccatgg agacagatac cctgctgctg tgggtgctgc tgctgtgggt ccctggcagc 60

accgga 66

<210> 26

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Gly Ser Ser Ser Ser Ser Ser Gly Ser Ser Ser Ser Ser Ser

1 5 10

<210> 27

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Gly Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser

1 5 10

<210> 28

<211> 24

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Ser

20

Claims

1. The fusion protein is characterized in that the fusion protein is formed by connecting an anti-human CTGF antibody and an extracellular section of a human type-II TGF-beta receptor through a joint, and the joint of the fusion protein is selected from (G) _x S _y ) _n A linker wherein x is 0, 1, 2, 3, 4, 5, y is 0, 1, 2, 3, 4, 5, n is 0, 1, 2, 3, 4, 5.

2. The fusion protein of claim 1, wherein the CDR of the light chain variable region of said anti-human CTGF antibody is selected from the group consisting of SEQ ID No.5, SEQ ID No.6, SEQ ID No.7, SEQ ID No.8, SEQ ID No.9, SEQ ID No.10; or the CDR of the heavy chain variable region of the anti-human CTGF antibody is selected from SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18; wherein the amino acid C in SEQ ID NO.15 can be substituted, preferably the amino acid C can be substituted by the amino acids S, Y, F.

3. The fusion protein of claim 2, wherein the light chain variable region of the anti-human CTGF antibody is selected from the group consisting of SEQ ID No.11 or SEQ ID No.12, or an amino acid sequence having at least 90% identity to SEQ ID No.11 or SEQ ID No. 12.

4. The fusion protein of claim 2, wherein the heavy chain variable region of said anti-human CTGF antibody is selected from SEQ ID No.19 or SEQ ID No.20, or an amino acid sequence having at least 90% identity to SEQ ID No.19 or SEQ ID No. 20.

5. The fusion protein of claim 1, wherein the constant region of the heavy chain of the anti-human CTGF antibody in the fusion protein is selected from the constant regions of human IgG1, igG2 and IgG4.

6. The fusion protein of claim 1, wherein the human type II TGF- β receptor has the sequence of SEQ ID No.2.

7. The fusion protein of claim 6, wherein the extracellular domain of the human type II TGF- β receptor of the fusion protein is selected from the group consisting of SEQ ID No.3 and SEQ ID No.4.

8. A biological vector, wherein the biological vector is a nucleic acid molecule encoding the fusion protein of any one of claims 1 to 7; the biological vector is an expression vector comprising the nucleic acid molecule as described above; the biological vector is a host cell comprising the aforementioned expression vector.

9. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 8 and/or the nucleic acid molecule of claim 8 and/or an expression vector and/or a host cell.

10. A method for preparing a fusion protein according to any one of claims 1 to 8, comprising the steps of:

(1) Splicing the light chain and the heavy chain of the antibody respectively;

(3) Separating and purifying after culturing the plasmid transformed cells.