CN113583142A - Double-target fusion protein, coding gene, vector or host cell and application and expression and purification method thereof - Google Patents
Double-target fusion protein, coding gene, vector or host cell and application and expression and purification method thereof Download PDFInfo
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- CN113583142A CN113583142A CN202110958223.9A CN202110958223A CN113583142A CN 113583142 A CN113583142 A CN 113583142A CN 202110958223 A CN202110958223 A CN 202110958223A CN 113583142 A CN113583142 A CN 113583142A
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Abstract
The invention provides a double-target fusion protein, an encoding gene, a vector or a host cell and an application, expression and purification method thereof, wherein the double-target fusion protein comprises an elastin-like double-function GLP-1 and FGF21 fusion protein. The protein is formed by fusing three fragments, wherein the first fragment comprises GLP-1 analogues, the second fragment comprises elastin-like proteins with different lengths, and the third fragment comprises FGF21 and analogues thereof, and the fragments are connected through a connecting peptide. The stability and the in vivo half-life period of the prepared novel fusion protein are obviously improved, and the novel fusion protein has better effects of reducing blood sugar and blood fat, controlling weight and improving liver injury on a model mouse. The fusion protein has better effect on treating metabolic diseases such as obesity, diabetes, hyperlipidemia, non-alcoholic fatty liver, atherosclerosis and the like.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to a double-target fusion protein, an encoding gene, a vector or a host cell and application and an expression and purification method thereof.
Background
Fibroblast growth factor 21(FGF21) is one of the FGF family members, a novel metabolic regulatory factor that acts specifically on liver, adipose and pancreatic islet tissue. Research shows that FGF21 has the functions of lowering blood sugar and blood fat, improving insulin resistance, protecting pancreatic island beta cell and other glycolipid metabolism regulation. Therefore, the compound has great potential in the clinical application of various metabolic diseases such as diabetes, obesity, atherosclerosis, fatty liver and the like. However, clinical trials find that FGF21 analogues do not have significant blood glucose improving effects in diabetic patients, which may be related to the activity and in vivo stability of designed FGF21 mutants, so that genetic engineering, functional fusion or chemical modification of FGF21 is becoming a hot point of research in recent years.
Glucagon-like peptide-1 (GLP-1) is a ghrelin produced by endocrine cells in the ileum. GLP-1 is expressed from the glucagon gene in 2 biologically active forms, GLP-1(7-37) and GLP-1(7-36) amides, differing in only one amino acid sequence, with about 80% of the circulating activity of GLP-1 arising from GLP-1(7-36) amides. GLP-1 helps to control food intake to reduce body weight, since it inhibits gastric emptying and reduces intestinal motility. And GLP-1 also has many other biological properties and functions, for example GLP-1 can play roles of reducing blood fat and blood pressure, thereby protecting cardiovascular system. GLP-1 is unstable in vivo and is easy to be hydrolyzed by DPP-4 enzyme, so that the enhancement of the stability and the activity of GLP-1 to enhance the drug effect has important significance in the field of biological pharmacy.
Elastin-like polypeptides (ELPs) are artificial polymers formed by connecting VPGXG pentapeptide repeat sequences in series, wherein X is other amino acid except Pro, have temperature-sensitive reversible phase transition characteristics, are soluble in a solution below the phase transition temperature and are condensed in a solution above the phase transition temperature, and the process is reversible. Therefore, the ELP fusion protein can be separated and purified by a simple centrifugation method, and the purification efficiency can be equivalent to that of affinity chromatography. In addition, since ELP is synthesized based on in vivo protein sequences, it has good biocompatibility and low immunogenicity, and has been widely used in the field of application of drug carriers.
FGF21 and GLP-1 exert the effect of regulating blood glucose and blood lipids through corresponding receptors on different target cells or different receptors on the same target cell. If the functions of the two can be effectively combined and generate a synergistic effect, the blood sugar can be controlled, the fat can be decomposed, and the weight can be reduced, so that the synergistic effect has obvious advantages compared with the single FGF21 or GLP-1 analogue in the treatment of metabolic diseases such as diabetes, obesity and the like.
Disclosure of Invention
Based on this, in order to solve the above technical problems, the present invention provides a double-target fusion protein, encoding gene, vector or host cell, and application and expression and purification method thereof, wherein the double-target fusion protein provided by the present invention is formed by connecting GLP-1 or its analogue and human FGF21 mutant through elastin-like protein (ELP), on one hand, the stability of the protein is improved, and on the other hand, the drug effect of the protein is increased. The preparation and production of the double-target fusion protein are realized by a recombinant DNA technology, and the double-target fusion protein has great potential in the treatment of diseases related to hyperglycemia and hyperlipidemia, such as diabetes, obesity, steatohepatitis or cardiovascular diseases, as a therapeutic drug or a pharmaceutical composition.
The invention provides a double-target fusion protein, which consists of the following fragments:
R1-R2-R3、R3-R2-R1、R1-L-R2-L-R3、R3-L-R2-L-R1；
wherein: r1 is human fibroblast growth factor 21(FGF21) or an analog thereof, R2 is an elastin-like protein, R3 is glucagon-like peptide 1(GLP-1) or an analog thereof; l is a connecting peptide.
In addition, the double-target fusion protein provided by the invention can also have the following additional technical characteristics:
further, the sequence of the R1 fragment is shown as SEQ ID NO: 1, or a sequence similar to SEQ ID No: 1, or a derivative protein having the same biological activity with a homology of 95% or more.
Further, the sequence of the R2 fragment is shown as SEQ ID NO: 2, or the sequence shown in SEQ ID No: 2 is increased or decreased to derive the protein.
Further, the sequence of the R3 fragment is shown as SEQ ID No: 3, or a protein corresponding to SEQ ID No: 3 has the same biological activity with the homology of more than 95 percent.
Further, the L-linker peptide consists of the amino acid sequence (GGGGS) n, where n may be an integer between 0 and 5.
Further, the pharmaceutically acceptable half-life prolonging mode is selected from the following components: polymers, unstructured (poly) peptide chains, serum proteins, serum protein binding molecules, antibodies, immunoglobulins, Fc regions/domains of immunoglobulins and immunoglobulin binding domains.
The invention also provides a gene carrying the coded amino acid sequence of the double-target fusion protein and a vector or a host cell thereof.
The invention also provides the application of the vector or the host cell in preparing a medicament for treating one or more diseases of diabetes, obesity, hepatitis or hepatitis related diseases.
The invention also provides a medicine or a medicine composition for treating metabolic diseases such as diabetes, obesity, non-alcoholic fatty liver and the like, hepatitis or related diseases, which comprises the double-target fusion protein.
The invention also provides an expression and purification method of the double-target fusion protein, which comprises the following steps:
construction of the gene expression vector carrying the double-target fusion protein: designing genes according to the codon preference of escherichia coli, connecting the synthesized target gene-containing fragment with a pET30a (+) vector, and transforming the escherichia coli to obtain an expression strain;
expression of the dual-target fusion protein: after the expression strain is cultured to a certain density, the expression of the double-target fusion protein is induced by IPTG, and the thalli are collected after the continuous culture for 4 to 10 hours;
and (3) purifying the double-target fusion protein: the obtained expression thallus is subjected to the working procedures of crushing, centrifugation, inclusion body denaturation, renaturation, ion exchange chromatography, ultrafiltration and the like, and finally the high-purity target double-target fusion protein is obtained.
Compared with the prior art, the invention has the beneficial effects that: (1) compared with single FGF21 and GLP-1 analogues, the double-target fusion protein has longer acting, more stable and better effects of treating obesity, overweight, metabolic syndrome, diabetes, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis, atherosclerosis, liver injury, liver cirrhosis, liver cancer, primary biliary cholangitis, primary sclerosing cholangitis and other metabolic diseases.
(2) The side effects of gastrointestinal discomfort and diet decline caused by the GLP-1 treatment process do not appear in the treatment process of the double-target fusion protein, and the influence on the normal life activities of organisms is small.
Drawings
FIG. 1 shows the results of electrophoretic analysis of the expression of GEF fusion proteins;
FIG. 2 is the results of liquid phase analysis of the purified GEF fusion protein;
FIG. 3 is an in vitro temperature stability assay for GEF fusion proteins;
FIG. 4 is an immunogenicity assay of the GEF fusion protein;
FIG. 5 shows the results of the weight effect of GEF fusion protein on HFD-induced NASH mice;
FIG. 6 shows the results of the effect of GEF fusion protein on blood glucose and OGTT in HFD-induced NASH mice;
FIG. 7 shows the results of the effect of GEF fusion protein on blood lipid and liver function in HFD-induced NASH mice;
FIG. 8 shows the effect of GEF fusion protein on the relative indices of HFD-induced NASH model mouse steatohepatitis and the like.
The following detailed description will further illustrate the invention in conjunction with the above-described figures.
Detailed Description
So that the manner in which the features and aspects of the invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments, some of which are illustrated in the appended drawings. The experimental procedures described in the following examples are only for demonstrating the feasibility of the present invention, and the application of the present invention is not limited thereto. The experimental procedures mentioned in the examples are, unless otherwise specified, conventional experimental procedures; the reagent consumables mentioned are conventional reagent consumables, unless otherwise specified.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.
The invention provides a novel medicine with the bifunctional bioactivity of human FGF21 and GLP-1.
In another aspect, the invention provides a gene encoded by the double-target fusion protein, a vector containing the gene sequence, and a host cell containing the vector.
The invention also provides a protein medicament for synergistically treating metabolic diseases by using the double-target fusion protein, which can be used for treating hyperglycemia, hyperlipidemia, hepatitis or related diseases such as diabetes, obesity, insulin resistance and the like, and can be used for reducing the weight of the liver and the content of triglyceride in the liver, repairing liver injury, inhibiting the expression of inflammatory factors, and improving the related metabolic syndromes such as non-alcoholic steatohepatitis, atherosclerosis, liver injury, liver cirrhosis, liver cancer primary biliary inflammation and/or primary sclerosing cholangitis.
In a first aspect of the invention, a dual-target fusion protein is provided comprising the amino acid sequences of FGF21 analogs, GLP-1 analogs, and elastin-like protein (ELP) and a linker peptide sequence of 0-30 amino acids between each sequence. The FGF21 amino acid sequence can be located at the C-terminus or at the N-terminus of the fusion protein. Preferably, FGF21 is located at the C-terminus of the fusion protein.
In a second aspect of the invention, there is provided an amino acid sequence, as shown in SEQ ID NO.4, which encodes a fusion protein of the invention as described above.
In a third aspect of the invention, there is provided an expression vector comprising the above DNA molecule.
In a fourth aspect of the invention, there is provided a host cell comprising the above-described expression vector.
In a fifth aspect of the invention, there is provided a method of producing a dual-target fusion protein of the invention, comprising the steps of:
culturing the host cell under the condition of expressing the fusion protein, thereby expressing the fusion protein, and purifying the process for preparing the fusion protein.
In a sixth aspect of the invention, there is provided a medicament and compositions thereof, comprising a pharmaceutically acceptable carrier or excipient or diluent, and comprising a fusion protein of the invention.
In a seventh aspect of the invention, a modification of the dual-target fusion protein is provided, which comprises a pharmaceutically acceptable half-life extending means selected from the group consisting of: polymers (e.g. polyethylene glycol (PEG), hydroxyethyl starch (HES), hyaluronic acid, polysialic acid), unstructured (poly) peptide chains (e.g. PAS, XTEN), serum proteins (e.g. albumin), serum protein binding molecules (e.g. Albumin Binding Domain (ABD), albumin binding fatty acids), antibodies, immunoglobulins, Fc regions/domains of immunoglobulins and immunoglobulin binding domains.
The following is an artificial sequence table of the double-target fusion protein of the invention:
example 1: construction, expression and purification of recombinant fusion protein
(1) Construction of fusion protein expression vectors
Designing a fusion protein GLP1-ELP-FGF21 Gene (GEF) according to the codon preference of escherichia coli, wherein the amino acid sequence of the gene is shown as SEQ ID NO: 4, respectively. The gene is synthesized by Nanjing Kingsrei company, and Nde I and BamH I enzyme cutting sites are designed at two ends of a target gene. The synthesized vector containing the target gene fragment and pET30a (+) are subjected to double enzyme digestion by Nde I and BamH I respectively, and after the enzyme digestion is finished, the required target fragments are recovered by glue. The fragment of interest was ligated with the prokaryotic expression vector pET30a (+) using T4-DNA ligase in a ligation system of 10. mu.L, mixed well, ligated overnight at 4 ℃ and then transformed into E.coli DH 5. alpha. each. And (3) selecting positive clones, and constructing a recombinant plasmid pET30a-GLP-ELP-FGF21(pET30a-GEF) after enzyme digestion identification.
(2) Expression and purification of fusion proteins
The recombinant plasmid pET30a-GLP-ELP-FGF21 containing the correct sequence was transformed into competent cells expressing the strain BL21(DE 3). Single colonies were picked and inoculated into 20mL LB medium containing Kan (50. mu.g/mL), respectively, cultured at 37 ℃ for 8 hours, inoculated into another 20mL LB medium containing Kan (50. mu.g/mL) at a volume ratio of 1:100, cultured at 37 ℃, when A600 was about 0.35, IPTG was added to a final concentration of 0.25mmol/L for induction, the induction temperature was 30 ℃, the cells were harvested after 5 hours, resuspended in PBS, disrupted, centrifuged, and the supernatant and the precipitate were taken, respectively, and analyzed by 12% SDS-PAGE electrophoresis. The results show that the GLP-ELP-FGF21(GEF) fusion protein is mostly expressed in the form of inclusion bodies in the Escherichia coli, and the expression amount is moderate (shown in figure 1).
Obtaining a large amount of induced thallus by high-density fermentation, enriching and washing the thallus by a 0.45 mu m-750kD hollow fiber membrane (20mmol/L Tris, 150mmol/L NaCl, pH8.0 buffer solution washing), adding lysozyme (1mg/mL) into the thallus, placing for 30min on ice, homogenizing for 2-3 times at 800bar high pressure with 700-fold ammonia water, until the thallus is completely broken. Enriching and washing the inclusion body by using a 750kD ultrafiltration hollow fiber column (3-5 times of pH 7.0-8.51M urea washing solution for washing and filtering); after the inclusion body is denatured and dissolved by using the urea denatured liquid with the pH value of 7.0-8.58M, the urea renaturation liquid with the pH value of 7.0-8.51M is diluted by 20-100 times, and finally the renaturation liquid is concentrated and replaced by buffer solution through a 5-10kD hollow fiber column to obtain the renaturated fusion protein.
Purifying the renaturated protein by a Q anion exchange chromatographic column, purifying the fusion protein by a step gradient elution mode, analyzing and identifying the obtained fraction by SDS-PAGE, finally purifying the target fraction by a 5-10kD hollow fiber column and replacing a buffer solution to obtain a pure protein, and performing high performance liquid chromatography analysis. The results showed that the purity of the liquid phase of the purified GEF fusion protein was more than 98% (as shown in FIG. 2).
Example 2: GEF fusion protein in vitro stability analysis
The purified GEF fusion protein was sterilized by filtration through a 0.22 μm filter and the protein was left at 37 ℃ for 72 hours. SDS-PAGE analysis shows that the GEF fusion protein does not generate obvious degradation bands after being placed at 37 ℃ for 72 hours (as shown in figure 3), which indicates that the GEF fusion protein has good in vitro stability.
Example 3: GEF fusion protein immunogenicity assays
24 male mice of SPF grade 8 weeks old were selected, randomly divided into a control group (Vehicle), an ELP-F group (elastin fusion FGF21), a GEF group and a G-ELP group (GLP-1 fusion elastin), and administered with the corresponding test substance to the experimental group once at about 8 o' clock each day in the morning, subcutaneously injected with a dose of 2mg/kg, the Vehicle group was injected with PBS of the same volume, administered continuously for 4 weeks, and after 4 weeks, the mice in each experimental group were sacrificed (fasting before night), and blood was sampled from eyeballs to measure the corresponding antibody to the recombinant protein in the serum of the experimental mice.
As shown in FIG. 4, the immunoreactivity of the Positive control group was significant, while the immunoreactivity was not detected in the ELP-F, EGF and G-ELP groups as in the Vehicle group, indicating that neither recombinant protein ELP-F, EGF nor G-ELP-injected mice were immunogenic.
Example 4: study of therapeutic effect of fusion protein on non-alcoholic steatohepatitis (NASH) model mouse
Experimental animals and breeding: the C57BL/6 mouse and animal experiments were performed in Jiangsu Jiejiaokang Biotech Co.
45 SPF-grade 6-week-old male C57BL/6 mice are fed with 60% High Fat Diet (HFD) for 4 months, abnormal body weight is eliminated, and 32 model mice with approximate mean values of blood sugar and body weight are screened and randomly divided into a Vehicle group, an ELP-F group, an EGF group and a G-ELP group, wherein each group comprises 8 mice. Another 8 same-week-old male C57BL/6 mice were used as a Normal control group (Normal group). The test substances are administered to the experimental group about 8.half a day in the morning, subcutaneously, 2mg/kg, and the Vehicle and Normal groups are injected with the same volume of physiological saline for 6 weeks. During the experiment, the patient can eat and drink water freely. During which time the mice were monitored for diet and body weight. 4 weeks after administration, fasting blood glucose levels (6h) and oral glucose tolerance (OGTT) were measured in individual experimental mice. After 6 weeks of administration, each experimental group of mice was sacrificed (fasting overnight), and liver levels of glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), total Cholesterol (CHOL), and low-density lipoprotein cholesterol (LDL-C) of the experimental mice were measured and tissue section staining and inflammation index detection were performed. The experimental data obtained were statistically analyzed.
As shown in FIG. 5, EGF and G-ELP both significantly reduced body weight of mice relative to saline control, while EGF mice controlled more significantly relative to G-ELP and ELP-F.
After 4 weeks of administration, fasting blood glucose and OGTT results of mice in each experimental group are shown in FIG. 6, compared with the normal saline group, fasting blood glucose levels of mice in the ELP-F group, the EGF group and the G-ELP group are remarkably reduced, oral glucose tolerance is remarkably improved, and simultaneously the blood glucose control capability of the EGF group is remarkably superior to that of the ELP-F group and the G-ELP group.
After 6 weeks of administration, the results of serum blood lipid and liver function parameters of mice in each experimental group are shown in fig. 7, compared with the normal saline group, the three protein groups can obviously reduce the levels of AST, ALT, CHOL and LDL-C in the serum of model mice, and the liver function and blood lipid levels of the EGF protein administration group are improved more obviously.
In addition, liver tissue sections of mice of each experimental group were subjected to HE staining and oil red staining, and the tissue sections were scored (from three aspects of steatosis, inflammatory lesions, and ballooning). The judgment standard is as follows: the sum is less than 3 points, namely non-NASH; the total score is more than 5 points, namely NASH is obtained; and 3 points < the total score < 5 points, namely the nondeterministic NASH. Pathological results show (see fig. 8, steatosis (black asterisk), inflammatory lesions (red small triangle), ballooning (blue small triangle), Bar 50 μm), Vehicle high-fat feeding group as a control group, Normal feed as a common feed, ELP-F group, EGF group, and G-ELP group as groups with obvious therapeutic effect on fatty liver, and GEF effect as the best, significantly reduced liver fat vacuole, and almost no vacuole observed under the liver pathological section microscope of EGF group.
According to the method and the results of the above examples, the EGF fusion protein prepared by the invention is discovered to have a treatment effect on the glycolipid flocculation and fatty liver symptoms of NASH diseases remarkably superior to that of the single ELP-F, G-ELP protein through a plurality of indexes of animal experiments, and the fusion protein has good treatment effect and application value in the treatment of metabolic diseases.
In summary, in the embodiments of the present invention, the dual-target fusion protein, the encoding gene, the vector or the host cell, and the application, the expression and the purification method thereof, compared with FGF21 and GLP-1 alone, the dual-target fusion protein of the present invention has the effects of longer-acting, more stable and better treatment of obesity, overweight, metabolic syndrome, diabetes, hyperglycemia, dyslipidemia, non-alcoholic steatohepatitis, atherosclerosis, liver injury, liver cirrhosis, liver cancer, primary biliary cholangitis, primary sclerosing cholangitis and other metabolic diseases; on the other hand, the double-target fusion protein has no side effect of gastrointestinal discomfort and diet decline caused by the GLP-1 treatment process in the treatment process, and has little influence on normal life activities of organisms.
The foregoing embodiments illustrate the principles, principal features and advantages of the present invention, and those skilled in the art will understand that the present invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the present invention and are not intended to limit the invention. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention. The present invention is subject to various changes and modifications without departing from the scope of the present invention, and such changes and modifications are intended to be included within the scope of the present invention.
Sequence listing
<110> innovative center of Jiangxiang Chinese medicine
<120> double-target fusion protein, encoding gene, vector or host cell and application and expression and purification method thereof
<130> 2021.8.20
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Asn Lys Ser Pro His Arg Asp Pro Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro 140
Gly Leu Pro Pro Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val 160
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Ser 181
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Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly 60
Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 80
Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly 100
Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly 120
Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly 140
Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly 160
Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly 180
Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly 200
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Gly Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 100
Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly 120
Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Gly Gly Val Pro Gly 140
Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly 160
Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly 180
Gly Gly Val Pro Gly Gly Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly 200
Val Gly Val Pro Gly Ala Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly 220
Val Gly Val Pro Gly Val Gly Val Pro Gly Gly Gly Val Pro Gly Gly Gly Val Pro Gly 240
Ala Gly Val Pro Gly Val Gly Val Pro Gly Val Gly Val Pro Gly Ala Gly Val Pro Gly 260
Val Gly Val Pro Gly Gly Gly Val Pro Gly Val Gly Gly Gly Gly Gly Ser Gly Gly Gly 280
Gly Ser Ala Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val Arg Gln 300
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Tyr Thr Ser 463
Claims (10)
1. A double-target fusion protein, which is characterized by consisting of the following fragments:
R1-R2-R3、R3-R2-R1、R1-L-R2-L-R3、R3-L-R2-L-R1；
wherein: r1 is human fibroblast growth factor 21(FGF21) or an analog thereof, R2 is an elastin-like protein, R3 is glucagon-like peptide 1(GLP-1) or an analog thereof; l is a connecting peptide.
2. The dual-target fusion protein of claim 1, wherein the sequence of the R1 fragment is as set forth in SEQ ID NO: 1, or a sequence similar to SEQ ID No: 1, or a derivative protein having the same biological activity with a homology of 95% or more.
3. The dual-target fusion protein of claim 1, wherein the sequence of the R2 fragment is as set forth in SEQ ID NO: 2, or the sequence shown in SEQ ID No: 2 is increased or decreased to derive the protein.
4. The dual-target fusion protein of claim 1, wherein the sequence of the R3 fragment is as set forth in SEQ ID No: 3, or a protein corresponding to SEQ ID No: 3 has the same biological activity with the homology of more than 95 percent.
5. The dual-target fusion protein of claim 1, wherein the L-linker peptide consists of the amino acid sequence (GGGGS) n, where n can be an integer between 0 and 5.
6. The dual-target fusion protein of claim 1, comprising a pharmaceutically acceptable half-life extending means selected from the group consisting of: polymers, unstructured (poly) peptide chains, serum proteins, serum protein binding molecules, antibodies, immunoglobulins, Fc regions/domains of immunoglobulins and immunoglobulin binding domains.
7. A gene encoded by the amino acid sequence carrying the double-target fusion protein of claim 1, and vectors or host cells thereof.
8. Use of a vector or host cell according to claim 7 in the manufacture of a medicament for the treatment of one or more of diabetes, obesity, hepatitis or hepatitis-related diseases.
9. A pharmaceutical agent or a pharmaceutical composition for treating metabolic diseases such as diabetes, obesity and non-alcoholic fatty liver disease, hepatitis or related diseases, which comprises the double-target fusion protein according to claim 1.
10. A method for expressing and purifying a double-target fusion protein comprises the following steps:
construction of the gene expression vector carrying the double-target fusion protein: designing genes according to the codon preference of escherichia coli, connecting the synthesized target gene-containing fragment with a pET30a (+) vector, and transforming the escherichia coli to obtain an expression strain;
expression of the dual-target fusion protein: after the expression strain is cultured to a certain density, the expression of the double-target fusion protein is induced by IPTG, and the thalli are collected after the continuous culture for 4 to 10 hours;
and (3) purifying the double-target fusion protein: the obtained expression thallus is subjected to the working procedures of crushing, centrifugation, inclusion body denaturation, renaturation, ion exchange chromatography, ultrafiltration and the like, and finally the high-purity target double-target fusion protein is obtained.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113735977A (en) * | 2020-05-28 | 2021-12-03 | 江苏康缘瑞翱生物医药科技有限公司 | rhFGF21 fusion protein, polynucleotide for coding same, composition containing same and application thereof |
| WO2024027553A1 (en) * | 2022-08-03 | 2024-02-08 | 无锡市华盛康肽生物技术有限公司 | Bifunctional fusion protein and use thereof |
| WO2024199481A1 (en) * | 2023-03-31 | 2024-10-03 | 上海多米瑞生物技术有限公司 | Protein analogue and use thereof |
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