CN117126262A - Recombinant variant mFGF21 protein, modified protein, and preparation methods and applications thereof - Google Patents
Recombinant variant mFGF21 protein, modified protein, and preparation methods and applications thereof Download PDFInfo
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- CN117126262A CN117126262A CN202310818715.7A CN202310818715A CN117126262A CN 117126262 A CN117126262 A CN 117126262A CN 202310818715 A CN202310818715 A CN 202310818715A CN 117126262 A CN117126262 A CN 117126262A
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Abstract
The invention provides a recombinant variant mFGF21 protein, a modified protein, and a preparation method and application thereof, belonging to the technical field of biological medicine. According to the invention, through the genetic engineering technology and the modification technology, the recombinant variant mFGF21 protein and the modified recombinant variant mFGF21 protein (PEG-mFGF 21) are obtained, animal and cell experiments show that the mFGF21 protein and the PEG-mFGF21 modified protein can obviously improve the lung function, reduce the lung weight ratio, reduce lung injury and collagen deposition, increase apoptosis and activate autophagy, thereby improving idiopathic pulmonary fibrosis, and the mFGF21 protein and the PEG-mFGF21 modified protein can be used as candidate therapeutic drugs for pulmonary fibrosis, and have the advantages of good safety, long duration of drug effect and better drug effect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a recombinant variant mFGF21 protein, a modified protein, a preparation method and application thereof, and further relates to application of the recombinant variant mFGF21 protein and the modified protein thereof in preparation of medicines for preventing, relieving and/or treating pulmonary fibrosis, in particular to application of the recombinant variant mFGF21 protein and the modified protein thereof in preparation of medicines for preventing, relieving and/or treating idiopathic pulmonary fibrosis.
Background
Idiopathic pulmonary fibrosis is a pulmonary disease characterized by fibroblast proliferation and extracellular matrix deposition. The american society of thoracic and european society of respiration redefine idiopathic pulmonary fibrosis, and indicate that idiopathic pulmonary fibrosis is a particular type of diffuse interstitial lung disease, an irreversible fibrosis of unknown cause, occurring in adult and limited to pulmonary and progressive fibrotic interstitial pneumonia. The disease of the idiopathic pulmonary fibrosis is progressive, the prognosis is poor, the death rate is high, about 60% of patients die because of the progress of the lung disease, the lung disease is dead to respiratory failure, and the median survival time is 2-3 years after the diagnosis of the idiopathic pulmonary fibrosis. In recent years, environmental pollution is increasingly serious, and the incidence rate of idiopathic pulmonary fibers is remarkably increased due to frequent weather such as haze and the like, so that serious threat is generated to life health of people. The aim of the treatment of idiopathic pulmonary fibrosis is to block disease progression, prolong survival, prevent acute attacks and alleviate symptoms. Current drug treatments for idiopathic pulmonary fibrosis, such as pirfenidone, nidanib recommended by the international guidelines of 2015. Pirfenidone reduces cell proliferation, production of fibrosis-related proteins, synthesis and aggregation of extracellular matrix, and reduces inflammatory cell aggregation caused by various stimuli; nidamib is a tyrosine kinase inhibitor that blocks intracellular signaling and inhibits proliferation, migration and transformation of fibroblasts. Clinical evidence shows that the two medicines have good curative effects on patients suffering from idiopathic pulmonary fibrosis, can delay the decline of pulmonary function and improve the quality of life of the patients, but have a plurality of adverse reactions, can not prevent the disease progression and can not prolong the life cycle of the patients. In addition, only lung transplantation surgery can reverse its course. Therefore, the development of the biological protein preparation with high safety and good curative effect aiming at idiopathic pulmonary fibrosis is the key point of treating pulmonary fibrosis at present.
Fibroblast growth factor 21 (FGF 21) is a novel member of the FGF family, a secretable protein. FGF21 is expressed mainly in tissues and organs such as liver, fat, skeletal muscle, pancreas, etc. FGF21 does not have fibroblast nutrition activity, has the main functions of regulating glycolipid metabolism, increasing insulin sensitivity, improving islet B cell function, reducing triacylglycerol level, improving obesity-related hyperglycemia and hyperlipidemia, reducing body mass and the like, and is closely related to metabolic diseases and cardiovascular diseases. FGF21 is safe and effective, does not produce hypoglycemia, causes side effects such as tumorigenesis and the like, and is expected to become a novel medicament for treating diabetes and related diseases. FGF-21 was developed in 2012 as a weight loss drug as well as a drug for treating diabetes, and this drug has entered the clinical trial stage. Scientists in 2013 reported that FGF21 has passed stage I clinic and that its safety was approved.
Related researches show that wild FGF21 inhibits pulmonary fibrosis by activating Nrf-2 pathway, but has the problems of poor inhibition effect and the like.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a recombinant variant mFGF21 protein, a modified protein, a preparation method and application thereof. The recombinant variant mFGF21 protein and the modified recombinant variant mFGF21 protein (PEG-mFGF 21) obtained by the genetic engineering technology and the modification technology have better curative effect in the idiopathic pulmonary fibrosis of mice, and the effect is obviously higher than that of wild type FGF21 (wt-FGF 21).
It is a first object of the present invention to provide a recombinant variant mFGF21 protein.
The amino acid sequence of the recombinant variant mFGF21 protein is shown as SEQ ID NO. 2.
Amino acid sequence of recombinant variant mFGF21 protein (SEQ ID NO: 2): ADSSPLLQFGGQVRQRYLYTDDARQTEAHLEIREDGTVGGAADQSPESLLQLRALAPGVIQILGVHTPRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNRSPHRDPAPRGPARFLPLPFLPPALPEPPGILGPQPPDVGSSDPLSMVGPSQGRSPSYAS
It is a second object of the present invention to provide a nucleic acid molecule encoding a recombinant variant mFGF21 protein as described above.
The nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.
Nucleotide sequence of recombinant variant mFGF21 protein (SEQ ID NO: 1): GCAGACTCCTCCCCACTGCTGCAATTTGGTGGTCAAGTACGTCAGCGTTACCTGTATACCGATGACGCGCGCCAGACCGAAGCCCACCTGGAAATTCGCGAAGATGGCACCGTTGGCGGTGCAGCTGACCAGTCTCCGGAATCCCTGCTGCAGCTGCGTGCGCTGGCCCCTGGTGTAATCCAGATCCTGGGTGTCCACACTCCGCGTTTCCTGTGTCAGCGTCCAGATGGCGCTCTGTACGGCTCTCTGCACTTCGACCCTGAAGCGTGCAGCTTTCGCGAGCTGCTGCTGGAGGATGGCTACAACGTTTACCAGTCCGAGGCACACGGTCTGCCGCTGCACCTGCCTGGCAACCGTTCTCCGCACCGCGACCCAGCCCCGCGTGGCCCGGCACGTTTTCTGCCTCTGCCGTTCCTGCCTCCAGCACTGCCGGAGCCGCCAGGTATTCTGGGCCCTCAGCCGCCTGACGTGGGCAGCTCTGATCCACTGTCCATGGTGGGCCCTTCCCAGGGCCGTTCCCCTTCCTACGCCTCT
A third object of the present invention is to provide a method for preparing a recombinant variant mFGF21 protein.
The preparation method comprises the following steps:
connecting a nucleic acid molecule shown as SEQ ID NO. 1 with an expression vector, transforming the nucleic acid molecule into a host cell, and carrying out fermentation treatment and crushing treatment to obtain a recombinant variant mFGF21 protein;
in some embodiments, the expression vector is pET30a or pET28a; the host cell is E.coli.
In some embodiments, the fermentation process comprises: the inoculation amount is 5-20%, the OD600nm value of induction is 5-10, the IPTG concentration is 0.5-2mM, the induction temperature is 18-37 ℃, and the induction time is 3-8h.
In the invention, the inventor optimizes the fermentation parameters and obviously improves the expression quantity of the target protein.
In some embodiments, the crushing treatment comprises: the fermented host cells are crushed using a high pressure homogenizer, in some preferred embodiments three times, in some more preferred embodiments the first crushing pressure is 500-600bar, and the second and third crushing pressures are 700-800bar.
In some preferred embodiments, the method of making further comprises the step of purifying the recombinant variant mFGF21 protein.
In some more preferred embodiments, the step of purifying the recombinant variant mFGF21 protein comprises: firstly, washing, denaturing and renaturation are carried out on inclusion bodies obtained after crushing treatment by adopting one or more of hollow fiber type, coiled type, tubular type or plate frame type membranes; and purifying the renatured sample by adopting an ion exchange column, and concentrating and replacing by using a buffer solution to obtain the recombinant variant mFGF21 protein with high purity.
Preferably, the membrane pore size used for said washing and said denaturing is 0.05-0.6. Mu.m, and the membrane pore size used for said renaturation is 3-10kD.
In the invention, the tangential flow membrane separation technology is adopted to wash, denature and renaturate the inclusion body, thus remarkably improving the yield of the target protein, and simultaneously purifying the target protein by utilizing an ion exchange column, thereby further improving the purity of the target protein.
It is a fourth object of the present invention to provide a modified recombinant variant mFGF21 protein.
The modified recombinant variant mFGF21 protein is a chemical modification product of the recombinant variant mFGF21 protein described above or the recombinant variant mFGF21 protein prepared by the preparation method described above.
A fifth object of the present invention is to provide a method for preparing a modified recombinant variant mFGF21 protein.
The preparation method comprises the following steps:
mixing the recombinant variant mFGF21 protein or the recombinant variant mFGF21 protein prepared by the preparation method with a PEG modifier for reaction to obtain a modified recombinant variant mFGF21 protein;
in some preferred embodiments, the temperature of the reaction is from 4 to 30 ℃ and the reaction time is from 2 to 24 hours;
in some preferred embodiments, the method of making further comprises the step of purifying the modified recombinant variant mFGF21 protein.
In some more preferred embodiments, the step of purifying the modified recombinant variant mFGF21 protein comprises: purifying the sample by using an ion exchange column, concentrating and replacing by using a buffer solution to obtain the modified recombinant variant mFGF21 protein with high purity.
In some embodiments, the molar ratio of the recombinant variant mFGF21 protein to the PEG modifier is 1:2-1:6, and in some preferred embodiments, the recombinant variant mFGF21 protein is at a final concentration of 8-12mg/ml in the mixed reaction system; and/or the number of the groups of groups,
the molecular weight of the PEG modifier is from 10 to 40kD, in some preferred embodiments, the molecular weight of the PEG modifier is 20kD; and/or the number of the groups of groups,
The PEG modifier is a polyethylene glycol aldehyde, and in some preferred embodiments, the PEG modifier is polyethylene glycol propionaldehyde.
According to the preparation method of the modified recombinant variant mFGF21 protein, the controllability of a modification process and a purification process of a modification target protein by a modifier can be improved, the yield and uniformity of the modification are ensured, and the yield of the target protein in the whole process are improved.
A sixth object of the present invention is to provide the use of the recombinant variant mFGF21 protein as described above, the recombinant variant mFGF21 protein produced by the production process as described above, the modified recombinant variant mFGF21 protein produced by the production process of any of the modified recombinant variant mFGF21 proteins as described above, for the preparation of a medicament for preventing, alleviating and/or treating pulmonary fibrosis disease, renal fibrosis disease, liver fibrosis disease, myocarditis disease, lipid metabolism disease, non-alcoholic fatty liver disease; in some preferred embodiments, the pulmonary fibrosis disease is idiopathic pulmonary fibrosis disease.
In some embodiments, the effective dose of the recombinant variant mFGF21 protein or the modified recombinant variant mFGF21 protein in the preventing, alleviating and/or treating is 0.1-10 mg/kg, the duration of the effect is 1-7 d; and/or the number of the groups of groups,
The route of administration of the recombinant variant mFGF21 protein or the modified recombinant variant mFGF21 protein in the preventing, alleviating and/or treating may comprise oral, intraperitoneal, subcutaneous, intravenous or intramuscular injection.
A seventh object of the present invention is to provide a medicament for preventing, alleviating and/or treating idiopathic pulmonary fibrosis.
In some embodiments, the active ingredient of the medicament comprises one or more of the recombinant variant mFGF21 proteins described above, recombinant variant mFGF21 proteins prepared by the preparation methods described above, modified recombinant variant mFGF21 proteins prepared by the preparation methods of modified recombinant variant mFGF21 proteins described above; and/or the number of the groups of groups,
the medicine also comprises a pharmaceutically acceptable carrier or auxiliary material; and/or the number of the groups of groups,
the route of administration of the drug includes oral, intraperitoneal, subcutaneous, intravenous or intramuscular injection.
An eighth object of the present invention is to provide the use of the nucleic acid molecule described above as a drug target in the screening of drugs for the treatment of idiopathic pulmonary fibrosis.
In some preferred embodiments, the agent is a small molecule activator of recombinant variant mFGF21 protein.
Compared with the prior art, the invention has the following beneficial effects:
1) The recombinant variant mFGF21 protein and the modified recombinant variant mFGF21 protein (PEG-mFGF 21) prepared by the invention have the characteristics of higher activity and better drug effect; compared with the wild type FGF21 protein, the same dosage of the mFGF21 protein and the modified recombinant variant mFGF21 protein has better effect of improving pulmonary fibrosis;
2) The invention develops a low-cost, high-expression and high-yield fermentation and purification system by optimizing fermentation conditions and combining tangential flow membrane separation and chromatography technology, and obtains high-purity recombinant variant mFGF21 protein; meanwhile, a fixed point modification technology is adopted, the protein modification efficiency is high, the problems of multiple modification and non-specific modification are solved, the quality of the obtained modified recombinant variant mFGF21 protein (PEG-mFGF 21) is stable and controllable, the overall preparation process stability is good, and stable process amplification can be realized;
3) The modified recombinant variant mFGF21 protein (PEG-mFGF 21) prepared by the invention has the characteristics of good safety, long duration of drug effect and the like, can obviously improve the progress of pulmonary fibrosis while reducing the administration frequency, and can inhibit collagen deposition, thereby having great significance for treating idiopathic pulmonary fibrosis.
Drawings
FIG. 1 is a graph showing the analysis of the expression level of a strain containing recombinant variant mFGF21 protein in example 1; wherein, lane 1: fermenting and collecting thalli; lane 2: supernatant after the thalli are crushed; lane 3: the bacterial cells are crushed and then deposited.
FIG. 2 shows SDS-PAGE (A) and HPLC analysis (B) of the recombinant variant mFGF21 protein after purification according to example 1 of the present invention.
FIG. 3 is a graph showing the effect of recombinant variant mFGF21 protein concentration and reaction time on PEG modification in example 2 of the present invention.
FIG. 4 is a graph showing the effect of the molar ratio of recombinant variant mFGF21 protein to modifier and the reaction temperature on PEG modification in example 2 of the present invention.
FIG. 5 is a graph showing the effect of pH of the reaction solution on PEG modification in example 2 of the present invention.
FIG. 6 shows SDS-PAGE (A) and HPLC analysis (B) of the purified PEG-mFGF21 protein according to example 2 of the present invention.
FIG. 7 is a graph showing the results of temperature stability studies of recombinant variant mFGF21 protein and PEG-mFGF21 protein according to example 3 of the present invention.
FIG. 8 is a graph showing the results of significantly improving BLM-induced lung function in mice using recombinant variant mFGF21 protein and PEG-mFGF21 protein according to example 4 of the present invention, wherein graph A is a graph showing the results of lung dynamic compliance (Cdyn); b is a Forced Vital Capacity (FVC) outcome graph; c is a graph of effort expiration time (FET) results; the meaning of the labels in the figures is as follows: in contrast to the set of models, * P＜0.05， ** P＜0.01， *** P＜0.001， **** P is less than 0.0001; in contrast to the group of wt-FGF21, # P＜0.05， ## P＜0.01， ### P＜0.001， #### P＜0.0001。
FIG. 9 is a graph showing the results of significantly reducing BLM-induced lung weight ratio of mice using recombinant variant mFGF21 protein and PEG-mFGF21 protein according to example 5 of the present invention, wherein graph A is a graph showing the results of lung dissection of mice; b, a lung dry-wet weight ratio result graph; the meaning of the labels in the figures is as follows: in contrast to the set of models, * P＜0.05， ** P＜0.01， *** P＜0.001， **** p is less than 0.0001; in contrast to the group of wt-FGF21, # P＜0.05， ## P＜0.01， ### P＜0.001， #### P＜0.0001。
FIG. 10 is a graph showing the results of the recombinant variant mFGF21 protein and PEG-mFGF21 protein of example 6 of the present invention for significantly improving BLM-induced pathological lesions and fibrosis in mice, wherein panel A is H&E, masson andimmunohistochemical staining (200X) results plot; panel B is a map of the results of Masson-stained collagen deposition analysis; panel C is a chart of the result of immunohistochemical staining analysis; d is a result graph of Hydroxyproline (HYP) content of the lung; the meaning of the labels in the figures is as follows: in contrast to the set of models, * P＜0.05， ** P＜0.01， *** P＜0.001， **** p is less than 0.0001; in contrast to the group of wt-FGF21, # P＜0.05， ## P＜0.01， ### P＜0.001， #### P＜0.0001。
FIG. 11 is a graph showing the results of the significant increase of TGF-beta induction of apoptosis of A549 cells by recombinant variant mFGF21 protein and PEG-mFGF21 protein according to example 7 of the present invention, wherein A is an apoptosis flow chart; b is a flow result statistical analysis result graph; the meaning of the labels in the figures is as follows: in contrast to the set of models, * P＜0.05， ** P＜0.01， *** P＜0.001， **** p is less than 0.0001; in contrast to the group of wt-FGF21, # P＜0.05， ## P＜0.01， ### P＜0.001， #### P＜0.0001。
FIG. 12 is a graph showing the results of significantly reducing TGF-beta induced epithelial mesenchymal transition of A549 cells by recombinant variant mFGF21 protein and PEG-mFGF21 protein according to example 8 of the present invention, wherein the symbols in the graph are as follows: in contrast to the set of models, * P＜0.05， ** P＜0.01， *** P＜0.001， **** p is less than 0.0001; in contrast to the group of wt-FGF21, # P＜0.05， ## P＜0.01， ### P＜0.001， #### P＜0.0001。
FIG. 13 is a graph showing the results of the activation of TGF-beta to induce autophagy of A549 cells by recombinant variant mFGF21 protein and PEG-mFGF21 protein according to example 8 of the present invention, wherein A is a transmission electron microscope (12000X); panel B is a graph of the results of statistical analysis of autophagosomes at the same fold; the meaning of the labels in the figures is as follows: in contrast to the set of models, * P＜0.05， ** P＜0.01， *** P＜0.001， **** p is less than 0.0001; in contrast to the group of wt-FGF21, # P＜0.05， ## P＜0.01， ### P＜0.001， #### P＜0.0001。
Detailed Description
The technical scheme of the present invention will be clearly and completely described in connection with specific embodiments. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
The experimental methods for which specific conditions are not specified in the examples are generally commercially available according to conventional conditions and those described in handbooks, or according to conditions recommended by the manufacturer, using general-purpose equipment, materials, reagents, etc., unless otherwise specified.
The amino acid sequence of the wt-FGF21 protein used in the examples was obtained in NCBI's GenBank: AAQ89444.1, and the gene sequence for translating the corresponding amino acid of the wt-FGF21 was ligated into pET30a (+) vector, and E.coli BL21 (DE 3) was transformed for expression, and purified to obtain the wt-FGF21 protein.
EXAMPLE 1 preparation of recombinant variant mFGF21 protein
1.1 construction and fermentation of recombinant variant mFGF21 protein-expressing Strain
The formula of the culture medium is as follows:
seed liquid medium (1L): angel (FP 108) peptone: 10g of Angel (FM 888) Yeast powder: 10g, K 2 HPO 4 ：1.74g，KH 2 PO 4 :1g, naCl:4g, 10mL of glycerol, pH 7.0.
Fermentation medium (4.5L): peptone: 50g, yeast powder: 50g, K 2 HPO 4 ：8.71g，KH 2 PO 4 :5g, naCl:20g, glycerol 50mL, and pH 7.0 adjusted with sodium hydroxide to a pH of 4.5L.
Feed medium (2L): 100g of peptone, 100g of yeast powder, adding distilled water to dissolve in a 1L volumetric flask as a nitrogen source, adding 665mL of glycerin into distilled water to dissolve in a 1L volumetric flask as a carbon source, and combining the two materials for sterilization.
(1) Construction of recombinant variant mFGF21 protein-expressing Strain
The sequence shown as SEQ ID NO is synthesized by Nanjing Jinsri company: 1, and two digestion sites of Nde I and Xho I are designed at two ends of the target gene. The synthesized fragment containing the target gene is inserted into a pET30a (+) expression vector and is transformed into escherichia coli DH5 alpha for amplification. And selecting positive clones, and performing enzyme digestion identification to construct the recombinant plasmid pET30a-mFGF21. The recombinant plasmid pET30a-mFGF21 containing the correct sequence is transformed into competent cells of the expression strain BL21 (DE 3). And selecting positive clone bacteria for expression identification to obtain a recombinant variant mFGF21 protein expression strain.
(2) Preparation of seed liquid
The recombinant variant mFGF21 protein-expressing strain was inoculated at an inoculum size of 1% into 20mL of seed culture medium containing 50. Mu.g/mL Kan, cultured at 37℃at 180rpm for 10.5 hours as a primary seed medium, then inoculated at an inoculum size of 1:100 into a shake flask containing 300mL of seed culture medium (containing 50. Mu.g/mL Kan), cultured at 37℃at 180rpm for 11 hours as a secondary seed medium, and the OD of the secondary seed medium was as that of the secondary seed medium 600 The value was 5.5.
(3) Fermentation treatment
Fermenting the secondary seed liquid, wherein the fermentation parameters are as follows: DO was controlled at 30% and pH at 7.00 before induction; DO was controlled at 20% after induction and pH at 6.95; and (3) material supplementing: 3h, feeding is started at the speed of: 8mL/min; the temperature before induction is 37 ℃, the induction temperature is 37 ℃ and the OD is induced 600 The value is 20+/-2, and the addition amount of the inducer is as follows: 2mL (1M IPTG), wherein the feeding speed is adjusted to 7mL/min when the fermentation speed is reduced to 900rpm for 6h, and the feeding speed is adjusted to 6mL/min when the fermentation speed is reduced to 800rpm for 7 h; after induction for 4-6h at 30 ℃, fermentation is finished, and the fermentation thalli are collected by centrifugation at 4000rpm for 10 min.
(4) Electrophoretic analysis of expression level of zymophyte protein
Taking 1mL of fermentation liquor, centrifuging at 12000rpm for 2min, discarding supernatant, adding 1mL of pure water, suspending and crushing; the horn No. 6, 40% power, 3s break 2s, 4min of work, 3 times break, the precipitate after centrifugation is suspended with 1mL pure water, 80. Mu.L of whole bacteria, supernatant and precipitate are taken for SDS-PAGE analysis, and the spotting amount is 5. Mu.L.
The result is shown in figure 1, and it can be seen from the figure that the target protein is mainly expressed in the form of inclusion bodies, and the yield of the target protein can reach more than 3g/L after high-density fermentation.
1.2 pretreatment of inclusion bodies
(1) Fermentation thallus disruption and inclusion body enrichment
Taking 300g of fermentation thalli, adding 0.15g of lysozyme and 15mg of deoxynuclease, re-suspending by using 3L of PBS buffer solution, incubating for 30min at 4 ℃, and crushing for three times by using a high-pressure homogenizer (the crushing pressure for the first time is 500-600bar, the crushing pressure for the second time is 700-800bar, and the temperature of a circulating water bath kettle is set to be 4 ℃).
And enriching inclusion bodies of the broken bacterial liquid by adopting a tube type centrifuge. Installing a condensing device of the tubular centrifuge, setting the rotating speed at 19500rpm and the sample injection speed at 50mL/min, enabling the bacterial liquid to be crushed to enter the centrifuge through a pipeline, continuously adding 1.5L PBS for centrifugation, collecting inclusion bodies after centrifugation is finished, weighing the wet weight of the inclusion bodies, and calculating the yield of the target protein enriched by the tubular centrifuge by using a gray scale method.
(2) Purification of inclusion bodies
1) Inclusion body wash
Taking 50g inclusion body after enrichment, re-suspending with 500mL PBS buffer, pouring the re-suspended sample into a tank of a membrane treatment system, and adopting 750kD hollow fiber column (model: UFP-750-E-8A; membrane area: 3600 cm) 2 ) Washing, in which the pump flow rate was set at 5.4L/min, the stirring speed of the tank solution was 40rpm, the inclusion bodies were washed with 2.5L of a washing solution (20 mM Tris+1-2M urea+0.1% Triton X-100, pH 8.0), the tank mass was maintained by changing the washing solution addition rate (the same applies hereinafter), and denaturation treatment was performed when 100mL of liquid remained in the apparatus (the volume of the sample collected at the permeation end was 2.9L).
2) Inclusion body denaturation
Closing a permeation end, adding 300mL of denatured solution (20 mM Tris+8M urea, pH 8.0), setting the flow rate to be 0.1-0.2L/min, opening the permeation end after circulation for 2h, collecting a denatured sample by the permeation end, gradually increasing the flow rate to be 5.4L/min, gradually adding 400mL of denatured solution into a tank until the volume of the collected sample is 300mL, continuously adding 600mL of renaturation solution until the total volume of the permeation end sample is 700mL, and stirring the permeation end solution in time until 100mL (the volume of the permeation end sample is 1.3L) of liquid remains in the instrument; 100mL of the retentate was discharged and the system was purged with pure water.
3) Inclusion body renaturation and buffer replacement
A10 kD hollow fiber column (model: UFP-10-C-8A; membrane area: 5300 cm) was used 2 ) Renaturation, concentration and buffer replacement are carried out on a denatured sample, the flow rate is set to be 2-3L/min, the pressure is increased to 0.7+/-0.02 bar, the volume of the denatured liquid is concentrated to 500mL, and 1.5L renaturation liquid (20mM Tris+0.02%Tween 80+10% glycerol, pH 8.0) is gradually added into a tank sample for renaturation treatment; then 1.5L of substitution solution (20 mM Tris+0.01% Tween 80, pH 8.0) was added to perform buffer substitution until the volume of the permeation end liquid became 3.8L, and the sample in the tank was discharged to obtain the intermediate renaturation protein.
1.3 ion exchange column purification of intermediate renaturation proteins
The intermediate renaturation protein was purified and separated by a Q Bestarose FF column (26X 100 mm) with a flow rate of 120cm/h, a mobile phase A of 20mM Tris+0.01% Tween 80 (pH 8.0), B of 20mM Tris+1M NaCl+0.01% Tween 80 (pH 8.0) and a detection wavelength of 280nm.
Firstly, setting 5% of mobile phase B for removing impurities, and stopping collecting fractions when the ultraviolet response value is 20mAU until the ultraviolet response value is reduced to 15 mAU; setting 20% of mobile phase B to elute target protein, starting to collect fractions when the ultraviolet response value is 20mAU, stopping collecting until the ultraviolet response value is reduced to 30-50mAU, and reserving a sample to be analyzed; finally, setting 100% of mobile phase B to remove the impurity protein with stronger retention, and starting to collect the fraction when the ultraviolet response value is 20mAU until the ultraviolet response value is reduced to 15mAU, and stopping collecting.
The purified sample was packed with ultrafiltration membranes (8-10 kD, membrane area: 0.11 m) 2 ) Concentrating and replacing buffer solution to obtain high-purity recombinant variant mFGF21 protein.
The result of SDS-PAGE electrophoresis analysis and SEC-HPLC analysis of the obtained recombinant variant mFGF21 protein is shown in figure 2, and the purified recombinant variant mFGF21 protein has the purity of more than 98 percent.
Example 2 preparation of PEG-mFGF21 protein
2.1 optimization of PEG modification conditions
(1) Optimization of modifier type and molecular weight
Preparing mother liquor of modifier (mPEG-pALD-20K, mPEG-pALD-40K, 2-arm PEG-CHO (LYS 01) -40K, Y-shape PEG-CH0 (T1 PT 02) - (8.6 x 2+2.8) K) with the concentration of 450mg/mL according to the following modifier: adding the recombinant variant mFGF21 protein into a recombinant variant mFGF21 protein sample according to the ratio of 4:1 to ensure that the final concentration of the recombinant variant mFGF21 protein is 10mg/mL, and then according to the reducing agent: reducing agent (sodium cyanoborohydride, 500 mg/mL) is added into the modifier according to the mol ratio of 10:1, and the mixture is placed at the temperature of 4 ℃ to react for 14-15h, so as to calculate the modification rate.
The calculated modification rate results are all above 85%, and the results show that the four modifying agents have no obvious difference in modification efficiency.
To further determine the PEG modifier species, the biological activity impact of the above four PEG modifiers on recombinant variant mFGF21 proteins was examined by cell and animal experiments. The result shows that the recombinant variant mPEG-pALD-20K modified mPFGF 21 protein has the highest activity retention rate of glucose absorption cells and better in vivo long-acting property.
(2) Optimization of recombinant variant mFGF21 protein concentration
According to the modifier: the modifier (mPEG-pALD-20K, 0.5 g/mL) was added to the 4 tubes (200. Mu.L per tube) containing recombinant variant mFGF21 protein samples at a protein molar ratio of 4:1, such that the final concentration of recombinant variant mFGF21 protein per tube was 6, 8, 10, 12mg/mL, respectively, and the pH was 6.5; and then according to the modifier: reducing agent (sodium cyanoborohydride, 0.5 g/mL) is added in the mol ratio of 1:10, the mixture is put into a water bath kettle at 25 ℃ for reaction, sampling is carried out respectively at the reaction time of 6h, 8h and 10h, and the protein modification rate is analyzed by electrophoresis.
The results are shown in FIG. 3, and it can be seen from the graph that there is no significant difference in the effect on the modification efficiency when the recombinant variant mFGF21 protein concentration is 8-12 mg/mL.
(3) Optimization of molar ratio of modifier to protein and reaction temperature
According to the modifier: the molar ratio of the proteins is 2:1, 4:1, and 6:1, respectively, the modifier (mPEG-pALD-20K, 0.5 g/mL) is added to 3 tubes (200 mu L per tube) of the recombinant variant mFGF21 protein-containing sample, so that the final concentration of the recombinant variant mFGF21 protein per tube is 6mg/mL and the pH is 6.5; and then according to the modifier: reducing agent (sodium cyanoborohydride, 0.5 g/mL) is added in the mol ratio of 1:10, the mixture is put into a water bath kettle at 25 ℃ for reaction, sampling is carried out respectively at the reaction time of 6h, 8h and 10h, and the protein modification rate is analyzed by electrophoresis.
Each group was modified according to the modifier: the modifier is added into 2 tubes (200 mu L of each tube) of recombinant variant mFGF21 protein-containing samples at a protein molar ratio of 4:1, so that the final concentration of each tube of recombinant variant mFGF21 protein is 10mg/mL and the pH is 6.5; and then according to the modifier: reducing agent (sodium cyanoborohydride, 0.5 g/mL) is added in the mol ratio of 1:10, and the mixture is respectively put into a water bath kettle at the temperature of 4 ℃ and the temperature of 25 ℃ to react for the same time, and the protein modification rate is analyzed by electrophoresis.
The results are shown in FIG. 4, from which it can be seen that when the mFGF21 protein concentration is 10mg/mL, the modifier: the mol ratio of the proteins is 6:1, and the modification effect is optimal when the reaction is carried out at 25 ℃ for 8 hours.
(4) Optimization of reaction pH
According to the modifier: the modifier is added into 3 tubes (200 mu L of each tube) of recombinant variant mFGF21 protein-containing samples respectively at the ratio of 4:1 of protein molar ratio, so that the final concentration of the recombinant variant mFGF21 protein in each tube is 6mg/mL, and the pH values are 6.5, 7.0 and 7.5 respectively; and then according to the modifier: 1 mu L of reducing agent (sodium cyanoborohydride, 0.5 g/mL) is added in the mol ratio of 1:10, and the mixture is put into a water bath kettle at 25 ℃ for reaction, and the protein modification rate is analyzed in the reaction for 6 hours, 8 hours and 10 hours.
As a result, it can be seen from FIG. 5 that the modification effect was optimal when the reaction pH was 6.5.
2.2 ion exchange column purification of PEG-mFGF21 protein
Modifying the recombinant variant mFGF21 protein by adopting optimized conditions (the final concentration of the mPEG-pALD-20K modifier and the recombinant variant mFGF21 protein concentration in a mixed reaction system is 10mg/mL, the molar ratio of the modifier to the protein is 6:1, the pH of the solution is 6.5, the reaction is carried out for 8 hours at 25 ℃), the PEG-mFGF21 protein is obtained, the PEG-mFGF21 protein is purified by adopting a Q Bestarose FF column (26 x 100 mm), the flow rate of the column is 120cm/h, the mobile phase A is 20mM Tris+0.01% Tween 80 (pH 8.0), the B is 20mM Tris+1M NaCl+0.01% Tween 80 (pH 8.0), and the detection wavelength is 280nm.
Firstly, setting 2% of mobile phase B for removing impurities, and stopping collecting fractions when the ultraviolet response value is 20mAU until the ultraviolet response value is reduced to 15 mAU; setting 10% of mobile phase B to elute modified protein, starting to collect fractions when the ultraviolet response value is 20mAU, stopping collecting until the ultraviolet response value is reduced to 30mAU, and reserving the samples to be analyzed; then, setting 18% of mobile phase B to elute the unmodified target protein, and starting to collect fractions when the ultraviolet response value is 20mAU until the ultraviolet response value is reduced to 30mAU, and stopping collecting; finally, setting 100% of mobile phase B to remove the impurity protein with stronger retention, and starting to collect the fraction when the ultraviolet response value is 20mAU until the ultraviolet response value is reduced to 50mAU, and stopping collecting.
The purified sample was packed with ultrafiltration membranes (8-10 kD, membrane area: 0.11 m) 2 ) Concentrating and replacing buffer solution to obtain high-purity PEG-mFGF21 protein.
The obtained PEG-mFGF21 protein is subjected to SDS-PAGE electrophoresis analysis and SEC-HPLC analysis, and the result is shown in figure 6, and the purified PEG-mFGF21 protein has the purity of more than 98 percent. The electrophoresis gray scale scanning analysis shows that the yield of the whole purification process of the target protein reaches more than 30 percent and the yield reaches 1g/L.
EXAMPLE 3 study of the temperature stability of recombinant variant mFGF21 protein and PEG-mFGF21 protein
The recombinant variant mFGF21 proteins and PEG-mFGF21 proteins obtained in example 1 and example 2 were placed at 37℃for 25 days, and samples were taken at 0, 5, 10, 15, 20, 25 days for SDS-PAGE analysis, respectively.
The results are shown in FIG. 7, and it can be seen from the graph that after the PEG-mFGF21 protein is incubated at 37 ℃ for 25 days, the purity can still be over 96%, the degradation rate is about 1.14%, and the degradation rate of the recombinant variant mFGF21 protein is about 20%, so that the results show that the in vitro temperature stability of the two proteins is better.
EXAMPLE 4 recombinant variant mFGF21 protein and PEG-mFGF21 protein to improve BLM-induced pulmonary function in mice
The recombinant variant mFGF21 proteins and PEG-mFGF21 proteins prepared in examples 1 and 2 were taken for use, and the wt-FGF21 protein was taken for use.
Experimental animals and feeding: 48 male C57BL/6 mice, 20-22g in weight, were purchased from Beijing vedorihua Biotechnology Co. All mice were placed under standard conditions (temperature: 22-24 ℃, humidity: 50-60%,12h light and dark cycle) and fed standard rodent diet. Mice were housed in an SPF-rated animal facility and after one week of adaptive feeding, the mice were randomly divided into 6 groups (8 per group):
(1) Normal group (PBS)
(2) Model group (BLM 3.5 mg/kg+PBS)
(3) QD group of wt-FGF21 2mg/kg (BLM 3.5mg/kg+wt-FGF21 2mg/kg daily treatment)
(4) mFGF21 2mg/kg QD group (BLM 3.5mg/kg+mFGF21 2mg/kg daily treatment)
(5) PEG-mFGF21 2mg/kg Q2D group (BLM 3.5mg/kg+PEG-mFGF21 2mg/kg day 2 treatment)
(6) PEG-mFGF21 4mg/kg Q4D group (BLM 3.5mg/kg + PEG-mFGF21 4mg/kg4 day treatment)
All groups were anesthetized with 2% sodium pentobarbital (intraperitoneal injection). A model of pulmonary fibrosis was established by intratracheal infusion of BLM (3.5 mg/kg) dissolved in sterile PBS, which was infused by normal group sham procedures. After 3 weeks, the normal and model groups were subcutaneously injected with PBS and the remaining groups were subcutaneously injected with different drugs. Mice were gas anesthetized 2d after the last dose. Lung function was measured prior to sacrifice using the Anires2005 system.
The results are shown in fig. 8, from which it can be seen that BLM-induced mice have significantly reduced lung compliance (Cdyn) and Forced Vital Capacity (FVC) while the forced breathing time (FET) is significantly increased, compared to normal mice. Compared with the model group, the mFGF21 protein can obviously improve the lung function, and compared with the wt-FGF21 group, the recombinant variant mFGF21 protein and the PEG-mFGF21 protein have more obvious capability of improving the lung function.
EXAMPLE 5 reduction of BLM-induced lung weight ratio of recombinant variant mFGF21 protein to PEG-mFGF21 protein in mice
The recombinant variant mFGF21 protein and PEG-mFGF21 protein obtained in example 1 and example 2 were taken for use, and the wt-FGF21 protein (obtained by expression purification preparation in the laboratory of the applicant)
The feeding and grouping of mice were identical to those in example 4.
The mice were anesthetized, spinal sacrificed, their lungs were isolated, washed with physiological saline, photographed and weighed.
The results are shown in fig. 9, from which it can be seen that BLM-induced mice had erosive scars and shrinkage of the lungs, while the lung weight ratio was significantly increased, compared to normal mice. Compared with a model group, the mFGF21 protein can obviously improve the lung erosive scar and reduce the lung weight ratio; compared with the group of the wt-FGF21, the recombinant variant mFGF21 protein and the PEG-mFGF21 protein can improve the lung erosive scar, and the capacity of reducing the lung weight ratio is more obvious.
EXAMPLE 6 recombinant variant mFGF21 protein and PEG-mFGF21 protein to alleviate BLM-induced pathological injury and fibrosis in mice lung
The recombinant variant mFGF21 proteins and PEG-mFGF21 proteins prepared in examples 1 and 2 were taken for use, and the wt-FGF21 protein was taken for use.
The feeding and grouping of mice were identical to those in example 4.
Mouse lung specimens were fixed with 4% paraformaldehyde, dehydrated, paraffin embedded and sectioned.
Paraffin tissue sections (5. Mu.M) were dewaxed and gradient ethanol hydrated and stained with hematoxylin, eosin (H & E) and Masson. In immunohistochemistry, after gradient hydration of the sections, the sections are soaked in 0.1mol/L citric acid antigen retrieval buffer solution, and antigen retrieval is carried out by a microwave method. Then, the sections were put into 3% hydrogen peroxide solution to block endogenous peroxidase, 3% BSA was added dropwise onto the tissue to cover the tissue uniformly, and the sections were incubated at room temperature for 1h to block nonspecific binding. The solution was washed off, primary antibody (. Alpha. -SMA) was instilled and the sections incubated overnight at 4 ℃. And (3) dripping a secondary antibody working solution, and incubating for 1h at room temperature. The freshly prepared DAB working solution was dropped onto a slide and the extent of staining was monitored under a microscope. Finally, the cells were counterstained with hematoxylin for 2min. Stained tissue sections were observed under a microscope.
As can be seen from the results in fig. 10, BLM-induced staining of mice lung H & E showed alveolar shrinkage, massive interstitial cell proliferation accompanied by inflammatory cell infiltration, massive collagen deposition in mouse lung tissue after Masson staining, and also massive proliferation of α -SMA marker myofibroblasts, myofibroblasts in model group lung tissue in immunohistochemical staining, and significant increase in collagen fiber content in model group in the Hydroxyproline (HYP) assay of total collagen fibers in lung. Compared with a model group, the recombinant variant mFGF21 protein can significantly improve the alveolar lesion and the pulmonary fibrosis of mice, compared with a wt-FGF21 group, the recombinant variant mFGF21 protein and the PEG-mFGF21 protein have more significant capability of improving the alveolar lesion and the pulmonary fibrosis of the mice, and the PEG-mFGF21 protein administered once in 2 days and once in 4 days has better drug effect, which indicates that the PEG-mFGF21 protein has better long-acting property.
EXAMPLE 7 recombinant variant mFGF21 protein and PEG-mFGF21 protein increasing TGF-beta to induce apoptosis of A549 cells
The recombinant variant mFGF21 proteins and PEG-mFGF21 proteins prepared in examples 1 and 2 were taken for use, and the wt-FGF21 protein was taken for use.
Human lung epithelial cells a549 cells were purchased from the Shanghai Qiao Xinzhou organism (lot number ZQ 0003).
RPMI 1640 medium, 1% penicillin-streptomycin, available from Solarbio; fetal bovine serum was purchased from ExCell corporation. Azoxymethane is available from sigma company (lot 098k 1488), dextran sodium sulfate MP biomeds (lot 0216011050); other medicines are made into domestic analytical pure. Human lung epithelial cell A549 cells were grown on the wall in RPMI 1640 medium containing 10% fetal bovine serum at 37℃with 5% CO 2 Culturing in a humidified incubator, and passaging once every other day.
Cells were grown to about 20% -30% of the cell dishes, and A549 cells were induced with 10. Mu.M TGF-beta for 72h modeling. Cells were then divided into 5 groups: blank group; a TGF-beta induced model group (TGF-beta); TGF-beta induced model group +2μM wild-type FGF21 (wt-FGF 21); TGF-beta induced model group +2μM recombinant variant FGF21 (mFGF 21); TGF-beta induced model group +2μM PEG modified FGF21 (PEG-mFGF 21), all drugs were allowed to act for 48h.
Cells were centrifuged after pancreatin digestion, 100 μl of 10 Xbuffer+2.5 μl of FITC stain+2.5 μl of PI stain was added to each tube, while 500 μl of PBS was added to the blank tube, and incubated at room temperature in the dark for 15min, after incubation was completed, 400 μl of Buffer was added to the test tube, and apoptosis was detected by flow cytometry.
The results are shown in FIG. 11, where it can be seen that TGF-beta induced A549 cells have significantly reduced apoptosis compared to normal A549 cells. Compared with the TGF-beta group, the apoptosis of cells can be obviously increased after the recombinant variant mFGF21 protein acts, and compared with the wt-FGF21 group, the recombinant variant mFGF21 protein and the PEG-mFGF21 protein prepared by the invention have more obvious capability of increasing the apoptosis of cells.
EXAMPLE 8 recombinant variant mFGF21 protein and PEG-mFGF21 protein reducing TGF-beta induced epithelial mesenchymal transition of A549 cells
The recombinant variant mFGF21 proteins and PEG-mFGF21 proteins prepared in examples 1 and 2 were taken for use, and the wt-FGF21 protein was taken for use.
Culturing and grouping of a549 cells were performed according to the method of example 7.
The level of the gene related to epithelial-mesenchymal transition in the A549 cells was detected by real-time quantitative q-PCR as follows:
(1) Preparing a solution:
1) 1mol/L Tris-HCl (pH 8.0): 121.1g of Tris is dissolved in 800mL of distilled water, the pH is adjusted to 8.0 by adding concentrated HCl, the volume is fixed to 1L, and the solution is sterilized under high pressure for standby.
2) 50×TAE buffer: 242g of Tris is dissolved in 500mL of double distilled water, 100mL of 0.5mol/L EDTA (pH 8.0) and 57.1mL of glacial acetic acid are added, the volume is fixed to 1L, and the mixture is preserved at room temperature for standby.
(2) The method comprises the following specific steps:
the general operation is to extract total RNA of the sample, then to detect the RNA, to detect the RNA by 1.2% electrophoresis gel, and to take 1 mu L RNA to detect by ultra micro spectrophotometer. UsingAll-in-One First-Strand cDNA Synthesis SuperMix (AE 341, TRANS) generates cDNA (reverse transcription). With quantitative PCR kit (/ -)>Green qPCR SuperMix) was quantified on a real-time PCR system in a 0.1mL flat-top eight-gauntlet premix reaction system. The fold change of mRNA expression level relative to the control group is obtained by adopting a 2-delta Ct method, and beta-actin is used as a control.
The beta-actin (beta-actin) internal reference primers were as follows:
F：5′-TGGCACCCAGCACAATGAA-3′(SEQ ID NO:3)，
R：5′-CTAAGTCATAGTCCGCCTAGAAGCA-3′(SEQ ID NO:4)；
the alpha smooth muscle actin (alpha-SMA) primers were as follows:
F：5′-GCGATCTCACCGACTACCTG-3′(SEQ ID NO:5)，
R：5′-GCCGACTCCATACCGATGAA-3′(SEQ ID NO:6)；
type I Collagen (Collagen I) primers were as follows:
F：5′-TGGCATCAAAGGACATCG-3′(SEQ ID NO:7)，
R：5′-CATAATACGGGGCAAAACC-3′(SEQ ID NO:8)。
the above primers were all synthesized by Shanghai Biotechnology Co., ltd.
The results are shown in FIG. 12, where it can be seen that TGF- β induces a549 cells with a significant increase in Collagen deposition (α -SMA/Collagen I) gene expression compared to normal A549 cells. Compared with the TGF-beta group, the recombinant variant mFGF21 protein can obviously reduce the collagen deposition of cells after being acted, and compared with the wt-FGF21 group, the recombinant variant mFGF21 protein and the PEG-mFGF21 protein have more obvious capability of reducing the collagen deposition of cells.
EXAMPLE 9 recombinant variant mFGF21 protein and PEG-mFGF21 protein increasing TGF-beta induction of A549 cell autophagosomes
The recombinant variant mFGF21 proteins and PEG-mFGF21 proteins prepared in examples 1 and 2 were taken for use, and the wt-FGF21 protein was taken for use.
Culturing and grouping of a549 cells were performed according to the method of example 7.
Then the following steps are carried out:
(1) Drawing materials and fixing: suspending cells/bacteria, centrifuging to collect cells or bacterial pellet, and requiring minimum pellet mung bean size. The medium was discarded, 2.5% glutaraldehyde was added, the cell mass was scattered with toothpick, and stored at 4 ℃. For adherent cells, the culture medium was poured off, 2.5% glutaraldehyde was added, the mixture was fixed at 4℃for 15min, scraped off with cells, collected by centrifugation (fixative retention), and stored at 4 ℃. Fixed time: at least 4 hours.
(2) Pre-embedding agar: centrifuging the cells or bacteria with a centrifuge, removing supernatant, adding 0.1M phosphate buffer (pH 7.4), mixing, rinsing for 3min, centrifuging, and repeating washing for 3 times. A1% agarose solution was prepared by heating and dissolving in advance, cooling slightly, then adding into an EP tube, and suspending and wrapping the precipitate in agarose with forceps before the agarose solidifies.
(3) Osmium acid fixation: cells fixed with glutaraldehyde were rinsed 3 times with 0.1M phosphate buffer (pH 7.4) for 15min each, and fixed with 1% osmium acid buffer (pH 7.4) at room temperature (20deg.C) for 2h (the fixing time was appropriately adjusted for each sample); then rinsed 3 more times with 0.1M phosphate buffer (pH 7.2) for 15min each.
(4) Dehydrating: the cells are dehydrated on 30%,50%,70%,80%,85%,90%,100% (twice) alcohol gradient for 15-20min each time (the water content is high, and the dehydration time of the cell film thickness sample can be prolonged appropriately). 100% acetone twice for 15min each.
(5) Penetration: acetone: 812 embedding medium = 1:1, penetration 2-4h at 37 ℃, acetone: 812 embedding medium = 1:2, permeation overnight at 37 ℃, pure 812 embedding medium permeation 5-8h at 37 ℃.
(6) Embedding: pure 812 embedding medium was poured into the embedding plate and the samples were inserted into the embedding plate and then oven overnight at 37 ℃.
(7) Polymerization: the embedding plate is put into an oven at 60 ℃ for polymerization for 48 hours, and the resin block is taken out for standby.
(8) Ultrathin section: the resin blocks are ultrathin sliced in an ultrathin slicer at 60-80nm, and a 150-mesh Fang Hua film copper net is fished.
(9) Double staining: dyeing the copper mesh in 2% uranium acetate saturated alcohol solution for 8min in a dark place; washing with 70% alcohol for 3 times; washing with ultrapure water for 3 times; 2.6% lead citrate solution is kept away from carbon dioxide for dyeing for 8min; the filter paper was slightly blotted by washing with ultrapure water 3 times. The copper mesh slices were placed in a copper mesh box and dried at room temperature overnight.
(10) Photographing and observing: and (5) observing under a transmission electron microscope, and collecting and analyzing images.
The results are shown in FIG. 13, where it can be seen that TGF-beta induced A549 cells have significantly reduced autophagy when compared to normal A549 cells. The recombinant variant mFGF21 protein of the present invention has a significantly increased autophagy after action compared to the TGF-beta group, and the recombinant variant mFGF21 protein and the PEG-mFGF21 protein of the present invention have a significantly increased autophagy compared to the wt-FGF21 group.
In summary, the invention develops a recombinant variant mFGF21 protein and a modified recombinant variant mFGF21 protein (PEG-mFGF 21), and in vivo and in vitro experimental researches show that the mFGF21 protein and the PEG-mFGF21 protein can improve the lung function of mice, reduce the lung weight ratio, reduce lung injury and collagen deposition, increase apoptosis and activate autophagy, thereby improving idiopathic pulmonary fibrosis. Compared with the wild type protein of the wt-FGF21, the mFGF21 and PEG-mFGF21 protein prepared by the invention have better drug effect in the aspect of treating the pulmonary fibrosis diseases of mice, and the PEG-mFGF21 protein has longer-acting and safer effects. Therefore, the mFGF21 and PEG-mFGF21 proteins have certain application prospects in the aspect of medicines for preventing, relieving and/or treating idiopathic pulmonary fibrosis.
The above examples are only specific embodiments of the present invention for illustrating the technical solution of the present invention, but not for limiting the scope of the present invention, and although the present invention has been described in detail with reference to the foregoing examples, it will be understood by those skilled in the art that the present invention is not limited thereto: any person skilled in the art may modify or easily conceive of the technical solution described in the foregoing embodiments, or perform equivalent substitution of some of the technical features, while remaining within the technical scope of the present disclosure; such modifications, changes or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention, and are intended to be included in the scope of the present invention.
Claims (10)
1. A recombinant variant mFGF21 protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 2.
2. A nucleic acid molecule encoding a recombinant variant mFGF21 protein according to claim 1, wherein the nucleotide sequence is as shown in SEQ ID No. 1.
3. A method for preparing a recombinant variant mFGF21 protein, comprising:
connecting a nucleic acid molecule shown as SEQ ID NO. 1 with an expression vector, transforming the nucleic acid molecule into a host cell, and carrying out fermentation treatment and crushing treatment to obtain a recombinant variant mFGF21 protein;
preferably, the preparation method further comprises a step of purifying the recombinant variant mFGF21 protein.
4. A modified recombinant variant mFGF21 protein, wherein the modified recombinant variant mFGF21 protein is a chemically modified product of the recombinant variant mFGF21 protein of claim 1 or the recombinant variant mFGF21 protein produced by the method of production of claim 3.
5. A method for preparing a modified recombinant variant mFGF21 protein, comprising:
mixing the recombinant variant mFGF21 protein of claim 1 or the recombinant variant mFGF21 protein prepared by the preparation method of claim 3 with a PEG modifier for reaction to obtain a modified recombinant variant mFGF21 protein;
Preferably, the temperature of the reaction is 4-30 ℃ and the reaction time is 2-24h;
preferably, the method of preparation further comprises the step of purifying the modified recombinant variant mFGF21 protein.
6. The method of claim 5, wherein the molar ratio of recombinant variant mFGF21 protein to PEG modifier is 1:2-1:6, preferably wherein the final concentration of recombinant variant mFGF21 protein in the mixed reaction system is 8-12mg/ml; and/or the number of the groups of groups,
the molecular weight of the PEG modifier is 10-40kD, preferably 20kD; and/or the number of the groups of groups,
the PEG modifier is polyethylene glycol aldehyde, preferably polyethylene glycol propionaldehyde.
7. Use of a recombinant variant mFGF21 protein according to claim 1, a recombinant variant mFGF21 protein prepared by the method of preparation according to claim 3, a modified recombinant variant mFGF21 protein according to claim 4, a modified recombinant variant mFGF21 protein prepared by the method of preparation of a modified recombinant variant mFGF21 protein according to claim 5 or 6, for the preparation of a medicament for preventing, alleviating and/or treating pulmonary fibrosis disease, renal fibrosis disease, liver fibrosis disease, myocarditis disease, lipid metabolism disease, non-alcoholic fatty liver disease; preferably, the pulmonary fibrosis disease is an idiopathic pulmonary fibrosis disease.
8. The use according to claim 7, wherein in said preventing, alleviating and/or treating the effective dose of said recombinant variant mFGF21 protein or said modified recombinant variant mFGF21 protein is 0.1-10 mg/kg and the duration of the effect is 1-7 d; and/or the number of the groups of groups,
the route of administration of the recombinant variant mFGF21 protein or the modified recombinant variant mFGF21 protein in the preventing, alleviating and/or treating may comprise oral, intraperitoneal, subcutaneous, intravenous or intramuscular injection.
9. A medicament for preventing, alleviating and/or treating idiopathic pulmonary fibrosis, characterized in that the active ingredient of the medicament comprises one or more of the recombinant variant mFGF21 protein of claim 1, the recombinant variant mFGF21 protein prepared by the preparation process of claim 3, the modified recombinant variant mFGF21 protein of claim 4, the modified recombinant variant mFGF21 protein prepared by the preparation process of claim 5 or 6; and/or the number of the groups of groups,
the medicine also comprises a pharmaceutically acceptable carrier or auxiliary material; and/or the number of the groups of groups,
the route of administration of the drug includes oral, intraperitoneal, subcutaneous, intravenous or intramuscular injection.
10. Use of the nucleic acid molecule of claim 2 as a drug target in the screening of drugs for the treatment of idiopathic pulmonary fibrosis.
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