Recombinant human ABD-IRISIN protein as well as preparation method and application thereof

Abstract

The invention relates to a recombinant human ABD-IRISIN protein as well as a preparation method and application thereof. The amino acid sequence of the recombinant human ABD-IRISIN protein provided by the invention comprises an amino acid sequence for coding an albumin binding domain ABD and an amino acid sequence for coding an IRISIN protein; the sequence for coding the albumin binding domain ABD and the sequence for coding the IRISIN protein are connected through a gene connecting element; and the gene connection element comprises a flexible connection gene element GS connexon. The problem that the half-life period of the IRISIN is short is solved, the IRISIN protein is modified through the albumin binding domain ABD, the recombinant human ABD-IRISIN protein which is the same as the natural IRISIN protein in structure and similar to biological activity and in-vivo pharmacokinetic characteristics is obtained, and compared with the unmodified IRISIN protein, the half-life period of the recombinant human ABD-IRISIN protein is prolonged by 20 times or above.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant human ABD-IRISIN protein and a preparation method and application thereof. Background Art

[0002] Irisin (IRISIN, Ir), as an exercise-related hormone discovered by Boström's team in 2012, has attracted widespread attention in the biomedical field in recent years. Studies have shown that IRISIN plays an important role in regulating a variety of physiological and pathological processes, including obesity, hypertension and cardiovascular disease. It can regulate mitochondrial energy metabolism, promote glucose utilization, enhance fatty acid oxidation and induce fat browning. In the complex pathological process of heart failure, IRISIN shows multi-dimensional effects on mitochondrial dysfunction, oxidative stress, metabolic imbalance, energy consumption and prognosis of heart failure. In addition, IRISIN is also widely present in tumor tissues and the serum of cancer patients, and its association with the occurrence and development of cancer has become a research hotspot. Many studies have confirmed that IRISIN can inhibit the proliferation, migration and invasion of tumor cells in vitro, participate in the inhibition of epithelial-mesenchymal transition (EMT) process, and affect the expression of key transcription factor Snail and E-cadherin gene transcription. More importantly, IRISIN shows potential application prospects in cancer prevention and treatment through mechanisms such as anti-inflammation, inducing cell apoptosis and enhancing the sensitivity of anti-tumor drugs.

[0003] Studies have shown that factors such as exercise can significantly stimulate the expression of skeletal muscle cell-induced transcriptional coactivator (PGC-1α). PGC-1α binds to the promoter region of the FNDC5 (fibronectin type III domain protein 5) gene to initiate its epitope translation and transcription. The synthesized FNDC5 reaches the endoplasmic reticulum under the action of the signal peptide, and then the signal peptide is cut off by the enzyme. The FNDC5 without the signal peptide completes N-glycosylation modification in the endoplasmic reticulum and Golgi apparatus and is transported out of the cell in the forward direction. After exocytosis, the transmembrane domain and the extracellular domain of FNDC5 are cut by proteases, and the extracellular domain forms irisin with biological function and enters the human circulation. The concentration of irisin in human blood is 3-4ng / mL, and its secretion can be increased under stimulation such as exercise and cold.

[0004] The half-life and physicochemical properties of irisin are determined by post-translational modifications, especially the effects of glycosylation. Glycosylation plays an important role in protein solubility, stability, structure, secretion and other cellular functions. In the human body, IRISIN is highly glycosylated. The Asn-36 and Asn-81 sites on the precursor FNDC5 of IRISIN are key sites for N-glycosylation. These glycosylation modifications contribute to the intracellular localization, secretion efficiency and half-life extension of IRISIN. However, despite the diverse biological functions of IRISIN, its extremely short in vivo half-life (less than 1 hour) greatly limits its potential application as a therapeutic agent or drug candidate.

[0005] Traditionally, IRISIN is modified by chemical modification to increase its half-life in vivo. However, the only modification method for irisin available on the market is to modify the N-terminal amino group of IRISIN with polyethylene glycol (PEG) and mutate the cysteine ​​at position 59 of IRISIN to serine, and perform prokaryotic expression and purification to solve the problem of irisin forming incorrect dimers during the renaturation process. For example: Chinese patent document CN113292645A discloses a PEG-modified irisin analog and a preparation method thereof. By using polyethylene glycol (PEG) for site-specific modification, the problem of irisin forming incorrect dimers during the renaturation process and poor stability in vivo is solved, but this also introduces new problems. Natural IRISIN exerts its biological effects through homodimers; in addition, it does not involve the detection of in vivo half-life, but only compares the stability of freeze-dried powder made of protein in a 60°C oven. Secondly, due to the heterogeneity of the size of PEG molecules, the use of PEG to modify proteins may result in different levels of PEG modification in each molecule in the modified protein population. This heterogeneous degree of modification may affect the functional properties and stability of the protein as well as its distribution and pharmacokinetic behavior in the body. In addition, in in vivo studies of proteins, PEG modification may also trigger immunogenic reactions, prompting the immune system to produce anti-PEG antibodies, affecting its biological activity in the body.

[0006] Therefore, it is of great significance to provide a more precise and controllable irisin modification method. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention aims to provide a recombinant human ABD-IRISIN protein, a preparation method and an application thereof, wherein the IRISIN protein is modified by the albumin binding domain ABD to prolong the half-life of the IRISIN protein, and the modified IRISIN is then expressed in large quantities in eukaryotic cells (including HEK-293T cells, HEK293F cells and Expi293 cells), so that the IRISIN can be fully modified, and a recombinant human ABD-IRISIN protein similar to the natural IRISIN protein is obtained.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions: The first object of the present invention is to provide a recombinant human ABD-IRISIN protein, the amino acid sequence of which comprises an amino acid sequence encoding an albumin binding domain ABD and an amino acid sequence encoding an IRISIN protein; the sequence encoding the albumin binding domain ABD and the sequence encoding the IRISIN protein are connected by a gene connection element; the gene connection element comprises a flexible connection gene element GS linker; The amino acid sequence encoding the albumin binding domain ABD is shown in SEQ ID NO.1; the amino acid sequence encoding the IRISIN protein is shown in SEQ ID NO.2; and the amino acid sequence of the recombinant human ABD-IRISIN protein is shown in SEQ ID NO.3.

[0009] GSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO.1); ADSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKE (SEQ ID NO. 2); GSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGSGSGSADSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKE (SEQ ID NO. 3).

[0010] The second object of the present invention is to provide a nucleic acid molecule encoding the above-mentioned recombinant human ABD-IRISIN protein.

[0011] Another object of the present invention is to provide a vector, which comprises the above-mentioned nucleic acid molecule.

[0012] Preferably, the vector is an exogenous plasmid.

[0013] Another object of the present invention is to provide a host cell, which comprises the above-mentioned vector.

[0014] Preferably, the host cell is a eukaryotic cell.

[0015] Preferably, the eukaryotic cells include HEK293T cells, HEK293F cells and Expi293 cells.

[0016] Another object of the present invention is to provide a method for preparing the above-mentioned recombinant human ABD-IRISIN protein, comprising the following steps: expressing the above-mentioned host cell, and then isolating and purifying it.

[0017] Another object of the present invention is to provide the use of the above-mentioned recombinant human ABD-IRISIN protein in the preparation of a product for prolonging the half-life of IRISIN in vivo.

[0018] Another object of the present invention is to provide the use of the above-mentioned recombinant human ABD-IRISIN protein in the preparation of a drug for treating lipopolysaccharide-induced inflammatory response.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention solves the problem of short half-life of IRISIN. The half-life of the prepared recombinant human ABD-IRISIN protein in vivo can reach nearly 11 hours, which is more than 20 times longer than the half-life of the unmodified IRISIN protein (only 0.5 hours). Moreover, at the same dose (500 μg / kg), the detection concentration of the recombinant human ABD-IRISIN protein provided by the present invention in the blood is 300 μg / mL, which is more than 1.6 times higher than the detection concentration of IRISIN protein in the blood of 180 μg / mL.

[0020] (2) The present invention modifies the IRISIN protein through the albumin binding domain ABD, and then expresses the modified IRISIN in large quantities in eukaryotic cells (including HEK-293T cells, HEK293F cells and Expi293 cells), so that the IRISIN can be fully modified, and a highly active recombinant human ABD-IRISIN protein similar to the natural IRISIN protein is obtained. The eukaryotic expression system overcomes the inherent disadvantage of the prokaryotic expression system of lacking post-translational modifications (such as glycosylation, phosphorylation, etc.), and the purified protein has high purity, showing similar biological activity and pharmacokinetic properties in vivo to the natural IRISIN protein.

[0021] (3) The recombinant human ABD-IRISIN protein provided by the present invention is used to prepare products for prolonging the half-life of IRISIN in vivo or to prepare drugs for treating lipopolysaccharide-induced inflammatory response, laying a solid foundation for subsequent scientific research and clinical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the structure diagram of the ABD-IRISIN gene; Figure 2 This is the plasmid map of pCDNA3.1(+)-ABD-IRISIN; Figure 3 is the plasmid map of pLenti-CMV-PURO-ABD-IRISIN; Figure 4 This is the positive control result of lentiviral transfection of pLenti-EGFP; Figure 5 This is the SDS-PAGE gel image of the nickel column affinity chromatography results; Figure 6 This is the SDS-PAGE gel image of the gel chromatography column filtration result; Figure 7 The SDS-PAGE gel images of purified IRISIN protein and recombinant human ABD-IRISIN protein; Figure 8 The results of incubation of purified recombinant human ABD-IRISIN protein with anti-IRISIN antibody and anti-His tag antibody respectively; Fig. 9 This is the result of Western Blot pulldown using His magnetic beads; Fig.10 The pictures are of Oil Red O staining of 3T3-L1 cells after induction of adipogenic differentiation and different treatment groups; Fig.11 The results of the microplate reader of the isopropanol eluate after Oil Red O staining in different treatment groups; Fig.12This is the standard curve for IRISIN concentration determination; Fig.13 The concentration trend of IRISIN protein and recombinant human ABD-IRISIN protein in the plasma of mice in the low-dose group (100 ng / g) over time; Fig.14 The concentration trend of IRISIN protein and recombinant human ABD-IRISIN protein in the plasma of mice in the medium-dose group (500 ng / g) over time; Fig.15 The concentration trend of IRISIN protein and recombinant human ABD-IRISIN protein in the plasma of mice in the high-dose group (1000 ng / g) over time; Fig.16 The change trend of IRISIN protein plasma concentration in mouse plasma over time; Fig.17 The change trend of the plasma concentration of recombinant human ABD-IRISIN protein in mouse plasma over time; Fig.18 The weight changes of each group in the LPS-induced inflammation mouse model; Fig.19 These are photos of the spleen tissue appearance and comparison of measurement values ​​in each group of the LPS-induced inflammatory mouse model. DETAILED DESCRIPTION

[0023] The above contents of the present invention are further described in detail below by means of specific implementation methods in the form of examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples.

[0024] Unless otherwise specified, the reagents used in the present invention are all conventional reagents, and the corresponding experimental operation methods and detection methods are all from conventional operation guides, such as the "Molecular Cloning Experiment Guide" and "Cell Experiment Guide" compiled by the Cold Spring Harbor Laboratory.

[0025] 1. Materials and equipment: The E. coli Stable strain used for plasmid cloning was purchased from Miaoling Biotechnology Co., Ltd.; the competent cells were made by our laboratory; The HEK-293F and Expi293 cell lines were purchased from Yonglian Biotechnology Co., Ltd. The mice were purchased from Zhejiang Weitonglihua Experimental Animal Technology Co., Ltd., model C57BL-6J; The endonucleases EcoRV and BamHI were purchased from Bio-Tech Biotechnology Co., Ltd., models D6337 and D6053; The nickel column was purchased from Boglon Biotechnology Co., Ltd., model Ni Bestarose FF, item number: AA0051; The SDL protein purification system was purchased from Suzhou Saipu Instrument Co., Ltd., model SCG-P; The IRISIN ELISA kit was purchased from Biotechnology Co., Ltd., catalog number: PI470.

[0026] 2. Design principle or inventive concept of the present invention: IRISIN protein, also known as irisin, is a protein secreted from muscles during exercise. It is an N-glycosylated protein composed of 112 amino acid residues with a molecular weight of about 32 kDa, and a molecular weight of about 20 kDa after deglycosylation (reference: [Boström P, Wu J, Jedrychowski MP, et al. A pgc1-α-dependentmyokine that drives brown-fat-like development of white fat and thermogenesis[J]. Nature, 2012, 481(7382): 463–468.]). IRISIN protein is a cleavage protein of fibronectin type III domain 5 (FNDC5), and its expression is controlled by peroxisome proliferator-activated receptor γ coactivator 1-α (PGC-1α). Although IRISIN protein is a protein with multiple biological activities, it has the functions of promoting fat browning and reducing lipid droplet formation, and plays an important role in energy metabolism, anti-inflammatory and immune system response. Existing studies have also shown its potential application prospects in the prevention and treatment of metabolic diseases such as obesity and diabetes. However, it also has some obvious shortcomings in practical applications. For example, the physiological mechanism of IRISIN protein is not fully understood; its extremely short half-life in vivo (less than 1 hour) greatly limits its potential application as a therapeutic agent or drug candidate.

[0027] Albumin is the most abundant plasma protein in the human body. In a healthy state, the half-life of albumin in adults is about 19 days. Binding drugs to albumin will affect the distribution, metabolism and therapeutic effects of these drugs, and eventually degrade into amino acids to provide nutrition for surrounding tissues and cells. In addition, the distribution of albumin in tumors and inflamed tissues increases, which can cause drugs to accumulate in inflamed tissues and tumor tissues. The albumin binding domain (ABD) originates from streptococcal protein G, has a molecular weight of only 5kDa, has a stable triple helical structure, is small in size, does not have any additional stabilizing features, and has a very high affinity with albumin.

[0028] The present invention modifies irisin (IRISIN protein) through an albumin binding domain (ABD). First, a gene sequence of a recombinant human ABD-IRISIN protein is designed and synthesized, and an ABD sequence is added to the N-terminus of IRISIN so that it can bind to albumin in vivo and prolong its half-life in vivo; secondly, a CD5 signal peptide or an IL-2 signal peptide is added so that ABD-IRISIN can be secreted and expressed; a 6×His tag is added to the N-terminus of IRISIN to facilitate purification; thirdly, the constructed ABD-IRISIN gene is inserted into a pLenti-CMV-PURO vector to construct an expression vector containing the recombinant human ABD-IRISIN protein gene sequence; then, the vector is transformed into a host cell (the host cell can use HEK293T cells, HEK293F cells or Expi293 cells) to induce expression, and a recombinant human ABD-IRISIN protein having the same structure as the natural IRISIN protein and similar biological activity and in vivo pharmacokinetic properties is separated from the expression product.

[0029] Example 1 Construction of ABD-IRISIN plasmid 1. Design and synthesize the gene sequence of recombinant human ABD-IRISIN protein By consulting the literature, the inventors of the present invention found that the integrity of the C-terminus of IRISIN plays an important role in its activity. In order to protect the C-terminus of the protein, all tags are added to the N-terminus, a 6×His tag is selected, and a signal peptide is designed to secrete and express the target protein. Codon optimization is performed according to the eukaryotic expression system, and the amino acid sequence information of the designed synthetic recombinant human ABD-IRISIN protein is shown in SEQ ID NO.6. Specifically: the CD5 signal peptide or the IL-2 signal peptide is added to the N-terminus of the protein, the 6×His tag is added after the signal peptide, and a TEV cleavage site is added after the His tag. After the TEV cleavage site are the ABD domain, the GS linker and the IRISIN amino acid sequence, wherein the amino acid information of the albumin binding domain ABD, IRISIN protein, signal peptide and TEV site are shown in SEQ ID NO.1~6, and the ABD-IRISIN gene structure diagram is shown in Figure 1 The amino acid sequence encoding the albumin binding domain ABD is shown in SEQ ID NO.1; the amino acid sequence encoding the IRISIN protein is shown in SEQ ID NO.2; the amino acid sequence of the recombinant human ABD-IRISIN protein is shown in SEQ ID NO.3; the amino acid sequence information of the signal peptide is shown in SEQ ID NO.4; the amino acid sequence information of the TEV cleavage site is shown in SEQ ID NO.5; the amino acid sequence information of the designed and synthesized recombinant human ABD-IRISIN protein is shown in SEQ ID NO.6.

[0030] MPMGSLQPLATLYLLGMLVASVLA(SEQ ID NO.4); ENLYFQG (SEQ ID NO. 5); MPMGSLQPLATLYLLGMLVASVLAHHHHHHENLYFQGGSLAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALPGSGSADSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKE (SEQ ID NO.6).

[0031] 2. Construction of the expression plasmid pCDNA3.1(+)-ABD-IRISIN containing the ABD-IRISIN gene The gene sequence of the recombinant human ABD-IRISIN protein was combined with the pCDNA3.1(+) plasmid using gene recombination technology to obtain the expression plasmid pCDNA3.1(+)-ABD-IRISIN containing the ABD-IRISIN gene. This process was entrusted to Sangon Biotech (Shanghai) Co., Ltd. The target fragment ABD-IRISIN was inserted into the pCDNA3.1(+) plasmid, and the restriction sites BamHI and EcoRV were inserted into the multiple cloning site region to obtain the expression plasmid pCDNA3.1(+)-ABD-IRISIN containing the ABD-IRISIN gene, wherein the plasmid map of the expression plasmid pCDNA3.1(+)-ABD-IRISIN is as follows Figure 2 shown.

[0032] 3. Acquisition of ABD-IRISIN target fragment The expression plasmid pCDNA3.1(+)-ABD-IRISIN containing the ABD-IRISIN gene is subjected to PCR amplification using primers containing the pLenti-CMV-PURO homology arm, followed by electrophoresis and gel recovery, and the product is recovered to obtain the ABD-IRISIN target fragment. The specific steps include: a) PCR amplification: The expression plasmid pCDNA3.1(+)-ABD-IRISIN containing the ABD-IRISIN gene was PCR amplified using primers containing the pLenti-CMV-PURO homology arms. The amplification system and PCR reaction conditions are as follows.

[0033] Table 4 Enzyme digestion system

[0034] The primers were commissioned to be synthesized by Sangon Biotech (Shanghai) Co., Ltd. The sequences are as follows: pLenti-CMV-PURO-ABD-IRISIN-F Primer Sequence Information: AAGACACCGACTCTAGATATCGCCACCATGCCCATGGGG (SEQ ID NO.7); pLenti-CMV-PURO-ABD-IRISIN-R Primer Sequence Information: TTTGTAGTCAGCCCGGGATCCTTACTCCTTCATGGTCACCTCGT (SEQ ID NO. 8).

[0035] Table 5 PCR reaction program

[0036] b) Agarose gel electrophoresis: The double-digestion products were electrophoresed on 1.0% agarose gel at 100 V / 40 min.

[0037] c) Recover the target band gel: After agarose gel electrophoresis, use a gel recovery kit (Conway Century, Product No.: CW2515S) to cut the gel and recover it. During recovery, repeatedly collect the melted gel solution on the adsorption column to recover the target band gel. After gel recovery, the recovered product ABD-IRISIN target fragment is obtained.

[0038] 4. Obtaining linearized fragments of pLenti-CMV-PURO vector The pLenti-CMV-PURO plasmid was double-digested with EcoRV and BamHI, and then electrophoresed and recovered by gel to obtain the linearized fragment of the pLenti-CMV-PURO vector. The specific steps include: a) Double digest the pLenti-CMV-PURO plasmid with restriction endonucleases EcoRV and BamHI. The quality system of the digested plasmid depends on the selected digestion system. Use 1ul of enzyme to correspond to 3ul of plasmid and incubate in a 37℃ water bath overnight.

[0039] Table 5 Enzyme digestion system

[0040] b) Agarose gel electrophoresis: The double-enzyme digestion products were electrophoresed on 0.8% agarose gel at 100V / 40min. Macroporous gel was used for agarose gel electrophoresis.

[0041] c) Recover the target band gel: After the agarose gel electrophoresis is completed, the gel is cut and recovered. During the recovery, the melted gel solution is re-collected on the adsorption column to recover the target band gel. After gel recovery, the linearized fragment of the pLenti-CMV-PURO vector is obtained.

[0042] 5. Connect The ABD-IRISIN target fragment obtained in step 3 and the pLenti-CMV-PURO vector linearized fragment obtained in step 4 are seamlessly connected using NEBuilder HiFi DNA Assembly recombinase to obtain a connection product. The specific steps include: The ABD-IRISIN target fragment and the pLenti-CMV-PURO vector linearized fragment were connected using NEBuilder HiFi DNA Assembly at 50°C for 15 minutes. The molar ratio of the ABD-IRISIN target fragment to the pLenti-CMV-PURO vector linearized fragment was 10:1.

[0043] Table 6 Connection system

[0044] 6. Obtain plasmid The ligation product was transformed into E. coli Sable competent cells, single clones were selected for amplification and first generation sequencing verification. The specific steps include the following: The ligation product of step 5 was transformed into Escherichia coli Stable competent cells, and the transformation product was spread on an LB solid culture plate containing 50 mg / L kanamycin. After growing at 37°C for 18 hours, a single colony was selected and transferred to an LB liquid culture medium containing 50 mg / L kanamycin, and shaken at 30°C at 300 rpm overnight. The plasmid was extracted according to the instructions of the plasmid extraction kit (Kangwei Century Endotoxin-free Plasmid Mini Extraction Kit), and named as pLenti-CMV-PURO-ABD-IRISIN plasmid.

[0045] The extracted plasmid was sent to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing identification. The ABD-IRISIN gene sequence is shown in SEQ ID NO.9, and the CMV promoter primer was used for first generation sequencing. The primer confirmed that the target gene ABD-IRISIN connection direction and sequence information were correct. Among them, the pLenti-CMV-PURO-ABD-IRISIN plasmid is shown in Figure 9. Figure 3 shown.

[0046] (SEQ ID NO.9).

[0047] CMV Promoter primer information is as follows: CGCAAATGGGCGGTAGGCGTG (SEQ ID NO. 10).

[0048] If the sequencing result is correct, the ABD-IRISIN plasmid can be obtained by expanding the culture and extracting the plasmid.

[0049] Example 2 Obtaining a cell line stably expressing ABD-IRISIN protein The ABD-IRISIN plasmid extracted in Example 1 was co-transfected with the lentiviral packaging plasmids PSPAX2 and pMD2G into eukaryotic cells (eukaryotic cells such as HEK293T cells, HEK293F cells and Expi293 cells) to obtain a lentiviral solution, and the lentivirus was used to infect eukaryotic cells to establish a stable cell line, and puromycin was used to screen positive cell lines. The culture was expanded, the supernatant was collected by centrifugation, and the solution was exchanged through a hollow fiber ultrafiltration system (BONA-GM-18 Shandong Bona Biotechnology Co., Ltd.). Specifically including: 1. Lentivirus packaging HEK-293T was used to package the target plasmid into lentivirus. The specific process is as follows: a) One day before transfection, the HEK293T cells grown in the T25 culture flask were digested and centrifuged, and the cells were resuspended in 12 mL of complete culture medium (DMEM medium containing 10% fetal bovine serum). 2 mL of cell suspension was added to each well of a six-well plate and cultured overnight in a wet culture medium at 37°C / 5% CO2.

[0050] b) When the cells grow to about 80%-90%, discard the overnight complete medium and replace it with serum-free and antibiotic-free medium (DMEM medium), 1 mL per well.

[0051] c) Prepare Solution A: Add 1.2ug of ABD-IRISIN plasmid (add 2.4μl if the plasmid concentration is 500ng / ul), 0.9μg of psPAX2 plasmid (add 1.8μl if the plasmid concentration is 500ng / ul), and pMD2.G plasmid (add 1.8μl if the plasmid concentration is 500ng / μl) to each well of a six-well plate, and prepare Solution A of ABD-IRISIN plasmid according to the volume of five wells; set up one well of positive control, using EGFP plasmid, to judge the transfection efficiency. (Target plasmid: psPAX2:pMD2.G=4:3:3).

[0052] d) Prepare Solution B: 100ul serum-free and antibiotic-free culture medium per well of a six-well plate, plus 25ul PEI. A total of 600ul DMEM + 125ul PEI for six wells.

[0053] e) Let solution A and B stand for 5 minutes, then add solution B drop by drop into solution A, flicking the tube while adding, and gently tap twice to mix well and let stand for 15-20 minutes. After standing, add the mixed solution evenly to a six-well plate and culture it in a wet culture sample at 37℃ / 5% CO2.

[0054] f) After three days of culture, observe the positive control wells under a fluorescence microscope. Figure 4 As shown, green fluorescence was observed in the cells, indicating that the lentivirus was successfully packaged.

[0055] g) Collect the lentiviral solution, sterilize it through a 0.22 μm filter membrane, and use it for transfection of HEK293T cells, HEK293F cells, or Expi293 cells, or freeze it in a -80°C refrigerator for storage.

[0056] 2. Lentivirus infection (taking HEK293F as an example) The lentivirus was infected into HEK-293F cells, and puromycin was added for selection until a stable cell line was successfully constructed. After the HEK293F-ABD-IRISIN stable cell line was successfully constructed, the cell line was expanded and cultured. The specific process is as follows: a) Take 5 OD cells of 293F with a viability of more than 95%, add 500 μl of lentiviral solution and 5 μl of polybrene, and culture at 37°C, 8% CO2 in an incubator at 180 rpm.

[0057] b) After 6-12 hours of culture, centrifuge the culture medium at 600×g for 5 minutes, discard the original culture medium, add new culture medium, resuspend to the original volume, and continue to culture in the incubator.

[0058] c) Three days later, puromycin was added to the culture medium at a final concentration of 2 ng / mL. After the cell viability began to increase, the puromycin concentration was gradually increased to 5 ng / mL.

[0059] d) After the cell viability is maintained above 90%, expand the culture.

[0060] 3. Collect samples and exchange solutions (taking Tris-HCl (pH=8.0) as an example) The cell supernatant was collected and centrifuged at high speed to remove cells and cell debris, and then the fusion was replaced with Tris-HCl (pH=8.0) solution using a hollow fiber ultrafiltration system (BONA-GM-18, Shandong Bona Biotechnology Co., Ltd.). The specific process is as follows: a) After adding protease inhibitors to the cell suspension, centrifuge the cell suspension at 3000×g for 15 min and collect the supernatant.

[0061] b) The supernatant after centrifugation was exchanged with Tris-HCl (pH = 8.0) using a hollow fiber ultrafiltration system (BONA-GM-18, Shandong Bona Biotechnology Co., Ltd.).

[0062] Example 3 Extraction and purification of protein The positive stable cell line obtained in Example 2 was expanded and cultured, the supernatant was collected by centrifugation, the solution was replaced by a hollow fiber ultrafiltration system (BONA-GM-18, Shandong Bona Biotechnology Co., Ltd.), and the purified ABD-IRISIN eukaryotic expression protein was obtained by nickel column affinity chromatography and gel chromatography column filtration. Specifically, it includes: 1. Affinity chromatography: The collected cell supernatant was subjected to affinity chromatography using a column, and eluted with 10mM imidazole elution solution and 200mM imidazole elution solution, respectively. 50mL 10mM imidazole was eluted twice, and 15mL 200mM imidazole was eluted three times. 15μl of the eluted liquid was sampled, 5μl 4× protein loading buffer was added, and heated in a 95℃ metal bath for 5 minutes to denature the protein; before loading, centrifuge, add 10~15ul to each well, select 5% stacking gel and 10% separation gel, set the electrophoresis instrument to 150V / 80min, and put the device in an ice bath. According to the SDS-PAGE bands, it was determined that the target protein was contained in the liquid eluted from the 200mM tube 1 and tube 2.

[0063] Table 7 10mM imidazole eluent composition (pH=8.0)

[0064] Table 8 200mM imidazole eluent composition (pH=8.0)

[0065] 2. Protein concentration: Use 3K ultrafiltration tube (PALL) at 2500rpm / 4℃ / 10min to concentrate the liquid in 200mM tube 1 and tube 2 to 2.5mL.

[0066] 3. Gel chromatography filtration: Prepare the eluent and use it after ultrasonic degassing. After loading the sample, observe and collect from the peak, and collect 3mL in each tube.

[0067] Table 9 Eluent (pH=8.0)

[0068] 4. SDS-PAGE electrophoresis: Take samples of the liquid collected in each tube and determine the peak corresponding to the target protein based on the SDS-PAGE bands.

[0069] a) Sample preparation: Take 15 μl of the collected liquid in each tube, add 5 μl loading buffer, heat in a 95℃ metal bath for 5 minutes, centrifuge instantly, and cool for later use.

[0070] b) Sample loading: Place the prepared gradient gel and gel plate device in the electrophoresis tank, make sure there is no leakage, pour in 1× electrophoresis buffer, slowly remove the comb, and add the sample and marker to the appropriate comb holes. Add 10~15μl to each hole, select 5% concentrated gel and 10% separation gel, set the electrophoresis instrument to 120V / 80min, and put the device in an ice bath.

[0071] Table 10 Preparation of SDS-PAGE polyacrylamide gel

[0072] c) Electrophoresis: Turn on the power supply, adjust the voltage to 120V, the time to 80min, start the voltage, and end the electrophoresis until the bromophenol blue runs to the bottom of the gel.

[0073] d) After electrophoresis, completely immerse the PAGE gel in Coomassie Brilliant Blue dye solution, heat in a microwave oven for 1 minute, and stain on a shaker for 10 minutes before discarding the dye solution. Wash with destaining solution 4 times, 30 minutes each time. Preliminarily judge the protein collection status based on the Coomassie Brilliant Blue staining results.

[0074] The same method as in Examples 1 to 3 was used to construct a plasmid expressing IRISIN, which was used to express and purify the IRISIN protein as a positive control for the biological activity experiment of the ABD-IRISIN recombinant protein.

[0075] 5. Analysis of experimental results: The experimental results are as follows Figures 5 to 8 As shown, by nickel column affinity chromatography, using different concentrations of imidazole gradient elution, such as Figure 5 As shown in the figure, when eluted with 200mM imidazole, the target protein band position (20-32kDa) was eluted, but there were obvious impurities, so the next step was gel chromatography column purification. The gel chromatography column was used to separate the protein, and the experimental results were as follows Figure 6 As shown, the protein elution was observed at the target band position. After collection, the protein was concentrated to further verify the protein purity. The experimental results are shown in Figure 7 As shown, the protein purity is above 95%, and anti-His tag antibody and anti-IRISIN antibody are used to verify whether it is the target protein. The results are as follows Figure 8 As shown, the purified recombinant protein has a His tag and an IRISIN protein sequence. In summary, according to the designed protein expression scheme, the ABD-IRISIN recombinant protein was successfully expressed and purified.

[0076] Example 4 Protein Verification After obtaining the target protein, the His-tagged protein was fixed using a nickel-containing magnetic column, and human serum albumin (HSA) was added to further explore the interaction between the target protein and human serum albumin (HSA).

[0077] 1. Western Blot Verification a) Use 5% stacking gel and 8% separation gel, load 20ug protein per well, and run electrophoresis at 80V for 30min, then at 120V until bromophenol blue reaches the bottom of the gel.

[0078] b) Take out the gel, select and cut the gel of appropriate width according to the needs, cut the PVDF membrane according to the size of the gel, activate it with methanol for 15 seconds, and then put it in the transfer solution for 5 minutes. Soak the sponge and thick filter paper with the transfer solution, and put the sponge, thick filter paper, PVDF membrane, gel, thick filter paper, and sponge on the side of the black electrode of the splint in turn. After installation, put it in the transfer tank and add transfer solution to cover the top of the transfer clip. Place the device in an ice bath and electrophoresis at 200mA for 90min.

[0079] c) Use TBST to prepare 5% skim milk powder as blocking solution. Place the membrane strips after electrophoresis in the blocking solution and incubate at 4°C overnight or at room temperature for 2 hours.

[0080] d) After blocking, wash the membrane 5 times with TBST, 5 minutes each time.

[0081] e) Anti-IRISIN rabbit monoclonal antibody was diluted in TBST to a concentration of 1:5000 and incubated overnight at 4°C.

[0082] f) After the primary antibody incubation, wash the membrane five times with TBST, 5 minutes each time.

[0083] g) HRP-labeled rabbit anti-mouse IgG antibody was diluted in TBST at a concentration of 1:8000 and incubated at room temperature for 2 hours.

[0084] h) After the secondary antibody incubation, wash the membrane five times with TBST, 5 minutes each time.

[0085] i) Mix the A solution and B solution of the ultrasensitive ECL chemiluminescence kit (the kit was purchased from Bio-Tech Biotech Co., Ltd., model P0018S) at a ratio of 1:1, and use 100ul of the mixed solution for each membrane strip. Add the mixed solution dropwise onto the membrane strip, mix well, incubate, and then expose immediately.

[0086] j) The size of the cell supernatant protein was detected to be about 28 kDa, which is the same as the theoretical value. (Reference: [Boström P, Wu J, Jedrychowski MP, et al. A pgc1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis [J]. Nature, 2012, 481 (7382): 463–468]. According to the above literature, after the post-translational glycosylation modification of IRISIN and the addition of the ABD domain, the theoretical size is about 28 kDa.) The experimental results are as follows Fig. 9As shown, the experimental results showed that in the pulldown group, only the ABD-IRISIN recombinant protein was able to specifically bind to HSA, while this interaction was not observed in the IRISIN protein control group, indicating that the ABD domain is intact and has biological functions.

[0087] 2. Cell Verification a) 3T3-L1 cells were cultured in a medium containing 0.5 mM IBMX, 1 μmol / L dexamethasone, and 10 μg / mL insulin for 2 days; then cultured in a medium containing only insulin for 2 days; and finally cultured in a conventional medium without induction hormones, with the medium changed every 2 days; 3T3-L1 cells were induced to become adipocytes.

[0088] b) After adding ABD-IRISIN recombinant protein and IRISIN protein to induced adipocytes in a six-well plate and culturing for 4 consecutive days, the culture medium was discarded, 500 μl of 4% paraformaldehyde was added to each well, and the cells were fixed in the dark for 2 h.

[0089] c) Photographs were taken after staining with Oil Red O and Hematoxylin.

[0090] d) Add 100 μl of isopropanol to each well of the six-well plate to elute Oil Red and measure the absorbance at 492 nM.

[0091] The experimental results are as follows Fig.10 As shown, the oil droplet staining in the group with IRISIN protein / ABD-IRISIN recombinant protein added was less than that in the control group with only inducer added.

[0092] In summary, the ABD-IRISIN recombinant protein purified and obtained by the present invention has the function of promoting fat browning and reducing the formation of lipid droplets, indicating that the protein has similar biological activity to IRISIN.

[0093] Example 5 Pharmacokinetics Testing After verifying that the protein has biological activity, the protein was injected into the mouse tail vein, and blood was collected from the mouse orbital venous plexus at different time periods to collect and prepare plasma. The IRISIN concentration in mouse plasma was detected using an IRISIN ELISA kit (Shanghai Biotech Co., Ltd., model PI470), as follows: a) Mouse dose grouping: C57BL-6 strain, 8-week-old mice were selected and two experimental cohorts were set up: ABD-IRISIN recombinant protein and IRISIN protein. Each cohort was divided into low, medium and high dose groups, with 3 mice in each group.

[0094] b) Blood was collected from mice in the control group and experimental group 30 minutes before injection, and blood was collected from the orbital venous plexus at 0.5h, 1h, 2h, 4h, 8h, 12h, and 24h after tail vein injection (the start of injection was counted as time 0), and 20μL of whole blood was collected and placed in a 1.5mL EP tube containing anticoagulant.

[0095] c) Centrifuge at 1000×g for 10 min at 4°C, then collect the plasma into new EP tubes and store at -20°C.

[0096] d) ELISA was used to measure the IRISIN concentration in mouse plasma.

[0097] The experimental results are as follows Figures 12 to 17 As shown, Fig.12 The results showed that R 2 =0.9993, indicating that the IRISIN ELISA concentration standard curve was successfully constructed. Fig.13 The results showed that the highest plasma concentrations of IRISIN and ABD-IRISIN in the low-dose group were 25.44 ng / g and 300.27 ng / g, respectively. The half-life of IRISIN was about 0.30 hours, and the half-life of ABD-IRISIN was about 9.03 hours. Fig.14 The results showed that the highest plasma IRISIN and ABD-IRISIN concentrations in the medium-dose group were 178.40 ng / g and 436.58 ng / g, respectively. The half-life of IRISIN in the medium-dose group was about 0.38 hours, and the half-life of ABD-IRISIN was about 13.46 hours. Fig.15 The results showed that the highest concentrations of IRISIN and ABD-IRISIN in the plasma of mice in the high-dose group were 282.26 ng / g and 656.12 ng / g, respectively. The half-life of IRISIN in the high-dose group was about 0.32 hours, and the half-life of ABD-IRISIN was about 11.51 hours. Fig.16 The results showed that the plasma IRISIN concentration in mice dropped rapidly after tail vein injection, and the half-life was less than one hour, which was consistent with the results reported in the literature. Fig.17 The results showed that the concentration of ABD-IRISIN in mouse plasma changed over time. When the fusion protein was used at low, medium and high doses, the half-life was about 9.03 to 13.46 hours, which was significantly better than the natural IRISIN protein. It can be seen that the half-life of the ABD-IRISIN recombinant protein of the present invention was extended by about 20 times in vivo compared with the IRISIN protein, and the plasma concentration was increased by more than 1.6 times.

[0098] Example 6 Inhibition of lipopolysaccharide (LPS)-induced inflammation model In order to further verify the role of the fusion protein designed in the present invention in inflammatory response, this study constructed an LPS-induced mouse inflammation model, and injected PBS, IRISIN protein, and ABD-IRISIN recombinant protein respectively to study the effect of ABD-IRISIN recombinant protein in regulating immunity, as follows: a) Mouse grouping: 24 wild-type C57BL-6 mice were selected, 3 mice in each group, and divided into 8 groups, namely PBS group (negative control group), LPS group (inflammatory model control group), LPS + IRISIN low, medium, and high dose groups, and LPS + ABD-IRISIN low, medium, and high dose groups. C57BL-6 strain, 8-week-old mice were selected, and two experimental cohorts of ABD-IRISIN and IRISIN were set up. Each cohort was set up with low, medium, and high dose groups, with 3 mice in each group.

[0099] b) Construction of LPS inflammation model: PBS group mice were intraperitoneally injected with 100μl PBS solution as negative control, and the other seven groups of mice were intraperitoneally injected with 100μl diluted LPS solution (concentration: 0.24 mg / mL). The frequency of administration was once a day. When the body temperature of the mice showed obvious changes (fever), the activity level of the mice decreased, and the amount of water and food consumed decreased; and orbital blood was taken from the inflammation model mice, inflammatory factors such as TNF-α and IL6 were tested by ELISA. When the inflammatory factors were significantly increased, it meant that the model was successfully constructed.

[0100] c) While using LPS to induce the inflammation model, the LPS group was injected with PBS through the tail vein as the inflammation model control group; the LPS + IRISIN group and the LPS + ABD-IRISIN low-, medium-, and high-dose groups were injected with different amounts of IRISIN (concentration: 0.12 mg / mL) and ABD-IRISIN (concentration: 0.12 mg / mL) through the tail vein, and the final concentrations were adjusted to 100 ng / g, 500 ng / g, and 1000 ng / g according to the weight of the mice. The frequency of administration was once a day.

[0101] d) Mouse body weight measurement: The mouse body weight was measured and recorded daily.

[0102] e) Mouse spleen tissue measurement: After 6 days of intervention, the mice were dissected and the spleen tissues were taken for photographing, measurement and recording.

[0103] f) H&E staining of major mouse organ tissues: The major organs of mice (heart, liver, spleen, lung, and kidney) were paraffin-embedded, sectioned, and H&E stained to evaluate the safety of ABD-IRISIN recombinant protein.

[0104] The experimental results are as follows Fig.18 and Fig.19 As shown. Fig.18 As shown in the figure, before administration, there was no difference in the weight of mice between the groups. As the number of days of administration gradually increased, the weight of mice in the LPS group gradually decreased compared with other groups, while the weight of mice in the LPS + IRISIN and LPS + ABD-IRISIN treatment groups at different concentrations had no significant difference from that in the PBS group. The spleen is an important immune organ, such as Fig.19 As shown in the figure, compared with the PBS group, the spleen of mice in the LPS group was significantly enlarged; compared with the LPS group, the spleen of mice in the LPS+Irisin and LPS+ABD-IRISIN groups was significantly reduced. Fig.19 As shown in (C), the spleen of mice in the treatment groups with different concentrations of IRISIN protein and ABD-IRISIN recombinant protein was reduced, but there was no significant difference. This suggests that the biological activities of ABD-IRISIN recombinant protein and IRISIN protein are equivalent, and both can alleviate the inflammatory response induced by LPS.

[0105] In summary, the present invention modifies the IRISIN protein by the albumin binding domain ABD, and then expresses the modified IRISIN in large quantities in eukaryotic cells (including HEK-293T cells, HEK293F cells and Expi293 cells), so that IRISIN can be fully modified, and a highly active recombinant human ABD-IRISIN protein similar to the natural IRISIN protein is obtained. The eukaryotic expression system overcomes the inherent shortcomings of the prokaryotic expression system, such as lack of post-translational modification (such as glycosylation, phosphorylation, etc.), and the purified protein has high purity, showing similar biological activity and pharmacokinetic properties in vivo to the natural IRISIN protein. Secondly, the present invention solves the problem of short half-life of IRISIN, and the half-life of the prepared recombinant human ABD-IRISIN protein in vivo can reach nearly 11h, which is more than 20 times longer than the half-life of the unmodified IRISIN protein, which is only 0.5h. Moreover, at the same dose (500 μg / kg), the detection concentration of the recombinant human ABD-IRISIN protein provided by the present invention in the blood is 300 μg / mL, which is more than 1.6 times higher than the detection concentration of the IRISIN protein in the blood is 180 μg / mL.

[0106] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A recombinant human ABD-IRISIN protein, characterized in that: Its amino acid sequence comprises an amino acid sequence encoding an albumin binding domain ABD and an amino acid sequence encoding an IRISIN protein; the sequence encoding the albumin binding domain ABD and the sequence encoding the IRISIN protein are connected by a gene connection element; the gene connection element comprises a flexible connection gene element GS linker; The amino acid sequence encoding the albumin binding domain ABD is shown in SEQ ID NO.1; the amino acid sequence encoding the IRISIN protein is shown in SEQ ID NO.2; and the amino acid sequence of the recombinant human ABD-IRISIN protein is shown in SEQ ID NO.

3.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the recombinant human ABD-IRISIN protein according to claim 1.

3. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 2.

4. The carrier according to claim 3, characterized in that The vector is an exogenous plasmid.

5. A host cell, characterized in that The host cell comprises the vector according to claim 3 or 4.

6. The host cell according to claim 5, characterized in that The host cell is a eukaryotic cell.

7. The host cell according to claim 6, characterized in that The eukaryotic cells include HEK293T cells, HEK293F cells and Expi293 cells.

8. A method for preparing the recombinant human ABD-IRISIN protein according to claim 1, characterized in that: The method comprises the following steps: expressing the host cell according to any one of claims 5 to 7, and then isolating and purifying the host cell.

9. Use of the recombinant human ABD-IRISIN protein according to claim 1 in the preparation of a product for prolonging the half-life of IRISIN in vivo.

10. Use of the recombinant human ABD-IRISIN protein according to claim 1 in the preparation of a drug for treating lipopolysaccharide-induced inflammatory response.

Images