CN113717269B

CN113717269B - Recombinant variant FGF21 protein and preparation method and application thereof

Info

Publication number: CN113717269B
Application number: CN202111014055.4A
Authority: CN
Inventors: 梁鑫淼; 叶贤龙; 郭志谋; 胡飞
Original assignee: Ganjiang Traditional Chinese Medicine Innovation Center
Current assignee: Ganjiang Traditional Chinese Medicine Innovation Center
Priority date: 2021-08-31
Filing date: 2021-08-31
Publication date: 2022-04-19
Anticipated expiration: 2041-08-31
Also published as: CN113717269A

Abstract

The invention discloses a recombinant human fibroblast growth factor 21 analogue (mFGF21) and a preparation method thereof. The recombinant mFGF21 protein coding gene is connected with an expression vector to obtain a recombinant plasmid, then a host cell is transformed, positive stable expression engineering bacteria are screened for high-density fermentation, the bacteria are centrifugally collected for high-pressure homogenization and crushing, and the high-density fermentation is subjected to the procedures of inclusion body enrichment, washing, denaturation, renaturation and buffer solution replacement, and the post-treatment of purification and membrane separation technology is carried out by adopting ion exchange chromatography to obtain the mFGF21 protein with the electrophoresis and liquid phase purity of more than 98%. The invention also discloses application of the recombinant mFGF21 protein in treating metabolic diseases such as obesity, diabetes, hyperlipidemia, non-alcoholic fatty liver disease and the like. Compared with wild hFGF21, the mFGF21 protein of the present invention has obviously raised stability and biological activity, and has obvious effect of improving blood sugar and blood fat level and fatty liver of model animal.

Description

Recombinant variant FGF21 protein and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a recombinant variant FGF21 protein and a preparation method and application thereof.

Background

With the increasing living standard, metabolic diseases represented by Diabetes Mellitus (DM) and non-alcoholic fatty liver disease (NASH) become invisible killers threatening human health. More than 1 hundred million people in insulin resistant diabetes patients (T2DM) in China can generate blindness, neuropathy, hypertension, liver and kidney pathological changes, cardiovascular diseases and other complications in the later period of onset, and the global market reaches 1200 hundred million dollars in 2025. Fatty liver has become a serious health threat to be seriously underestimated, the number of patients exceeds that of diabetes (T2DM 1 billion), and the fatty liver can cause cardiovascular and metabolic diseases or accelerate the disease process besides causing serious liver diseases and even canceration. Nearly 4.3 million people in China suffer from non-alcoholic fatty liver disease, the global prevalence rate is up to 25 percent, more than 17.5 million people, and the global market reaches 400 million dollars in 2025 years.

If DM disease cannot be effectively controlled, multiple system damages in vivo can be caused, which causes chronic progressive lesions of tissues such as eyes, kidneys, nerves, heart, blood vessels and the like, and causes functional defects or failure. The characteristics of diabetes with a plurality of complications and high disability rate become public health problems needing to be addressed worldwide at present. At present, no cure method exists clinically, and patients need to take medicines for life to maintain stable blood sugar. Also, the risk of hepatic fibrosis and liver cancer will be significantly increased in NASH diseases such as those in the late stage of non-treatment, and no related drugs are currently approved for marketing in the treatment of the disease.

Fibroblast growth factor (FGF21) is a new member of fibroblast growth factor family, and has high homology between its amino acid sequence and member of FGF19 subfamily. A large number of experiments show that FGF21 is another regulating factor capable of independently regulating blood sugar and blood fat in vivo, can promote 3T3-L1 fat cells to consume glucose in vitro, has the functions of reducing blood sugar level, reducing triglyceride, increasing high-density cholesterol lipoprotein, reducing low-density cholesterol lipoprotein and the like in vivo, and does not have the side effects of hypoglycemia, hyperinsulinemia, weight gain, edema and the like. With the progress of research, researchers find that FGF-21 not only has an important regulation effect on glycolipid metabolism, but also has functions related to the aspects of improving pancreatic beta cell function, delaying the generation of chemically induced liver cancer, reducing body weight, reducing fat accumulation and the like. These characteristics endow FGF21 with the prerequisite for new drugs for treating metabolic syndrome with abnormal glycolipid metabolism, and FGF21 as a safe and reliable metabolic regulation factor has great potential to become a new drug for improving insulin resistance. Therefore, FGF21 has important significance in developing drugs for treating various metabolic diseases such as diabetes, obesity, non-alcoholic fatty liver disease and the like. However, clinical trials find that FGF21 has no significant effect on improving blood glucose in diabetic patients, which may be related to the biological activity and in vivo stability of designed FGF21 mutant, so genetic engineering and chemical modification of FGF21 are becoming the focus of research in recent years.

Disclosure of Invention

Therefore, the variant mFGF21 protein is designed and constructed through computer-aided prediction and experiments, and the mFGF21 protein with good stability and biological activity is prepared through optimized expression and purification processes. Animal experiment results show that the mFGF21 protein has good efficacy on obesity, diabetes and NASH model mice, and the treatment effect is remarkably superior to that of wild-type FGF21(hFGF 21).

The invention aims to provide a novel variant mFGF21 protein, the amino acid sequence of which is shown as SEQ ID NO. 2; or sequences having high homology to the above sequences (e.g., at least 95%, at least 98%, at least 99% homology, or having one or more amino acid substitutions (e.g., conservative substitutions), deletions, and/or additions); or at least one amino acid mutation at position 56 or 59 or 69 or 122, wherein the amino acid mutation at position 56 is selected from any one of the following amino acid mutations: K56R; K56A; K56H; K56G; K56L; K56I, amino acid mutation at position 59 selected from any one of: K59R; K59A; K59H; K59L; K59I, the amino acid mutation at position 69 is selected from any one of the following amino acid mutations: K69R; K69A; K69H; K69L; K69I, wherein the 122 th amino acid mutation is selected from any one of the following amino acid mutations: K122R; K122A; K122H; K122L; K122I.

Another objective of the invention is to provide a DNA sequence for encoding the mFGF21 protein, a vector containing the DNA sequence and a host cell containing the vector.

Further, the preparation method of the variant mFGF21 protein comprises the following steps:

the method comprises the following steps: construction of a gene expression vector carrying a target protein: the synthesized gene segment of SEQ ID NO. 1 in the sequence table is connected with a prokaryotic expression vector to transform escherichia coli, and an expression strain is obtained.

Step two: expression of mFGF21 protein: culturing the recombinant expression strain and inducing the recombinant expression strain to produce the target protein;

step three: purification of mFGF21 protein: and (4) harvesting the expression strain treated in the step two, and finally obtaining the high-purity target protein through the working procedures of crushing, centrifuging, membrane separation, ion exchange chromatography, ultrafiltration and the like.

Furthermore, the invention also provides a pharmaceutical composition containing the mFGF21 protein for treating metabolic diseases, which can be used for treating diabetes, obesity, non-alcoholic fatty liver or related diseases, including reduction of liver weight and content of liver triglyceride, repair of liver injury, and improvement of metabolic syndromes related to non-alcoholic steatohepatitis, liver injury, liver cirrhosis and the like.

In still another aspect, the invention also provides the use of the pharmaceutical composition in the preparation of a medicament for preventing or treating metabolic diseases.

Furthermore, the invention also discloses a modified product, which is obtained by modifying mFGF21 protein by adopting a pharmaceutically acceptable chemical modifier, wherein the chemical modifier is selected from one or more of the following substances: polymers (e.g. polyethylene glycol (PEG), hydroxyethyl starch (HES), hyaluronic acid, polysialic acid), unstructured (poly) peptide chains (e.g. PAS, XTEN), elastin-like polypeptides (ELP), serum proteins (e.g. albumin), serum protein binding molecules (e.g. Albumin Binding Domain (ABD), albumin binding fatty acids), antibodies, immunoglobulins, Fc regions/domains of immunoglobulins and immunoglobulin binding domains.

The preparation of the variant FGF21 protein is preferably carried out by using an escherichia coli expression system, and an integral purification process with high expression, high yield and low cost is developed by combining membrane separation and chromatographic techniques, so that the high-purity target protein can be stably and efficiently prepared.

The invention has the beneficial effects that:

(1) compared with wild hFGF21, the variant FGF21 protein has the effects of being long-acting, stable and better in treating obesity, diabetes, hyperglycemia, dyslipidemia, nonalcoholic steatohepatitis (NASH), atherosclerosis, liver injury, liver cirrhosis, liver cancer and other metabolic diseases.

(2) The invention provides a high-efficiency and stable integral purification method of FGF21 protein. Compared with a eukaryotic expression system (the expression amount is about 30-70mg/L), the invention adopts an escherichia coli system and an adaptive expression vector to obtain higher expression amount, thereby obviously reducing the production cost; and the integral purification process developed by the integrated membrane separation and chromatographic separation technology is simple and convenient and has high yield, and the method has the advantages of stability, easiness in linear method, high yield and the like, and lays a solid foundation for industrial research of protein medicines in later period.

Drawings

FIG. 1 is a diagram of the expression identification analysis of hFGF21 and mFGF21 proteins of the present invention in E.coli;

FIG. 2 is a diagram showing the electrophoretic and high performance liquid chromatography analyses of the purified mFGF21 protein of the present invention;

FIG. 3 shows the plasma incubation stability and in vivo half-life of mFGF21 protein of the present invention;

FIG. 4 is a graph showing the in vitro cell activity assay of mFGF21 protein of the present invention;

FIG. 5 is a graph showing the effect of the mFGF21 protein of the present invention in regulating blood glucose levels in HFHC feed-induced NASH model mice;

FIG. 6 is a graph showing the effect of mFGF21 protein of the present invention on controlling the body weight of mice in HFHC feed-induced NASH model;

FIG. 7 is a graph showing the effect of mFGF21 protein of the present invention on improving blood lipid levels in HFHC feed-induced NASH model mice;

FIG. 8 is a graph showing the effect of mFGF21 protein of the present invention on the improvement of HFHC feed-induced NASH model mouse liver injury;

FIG. 9 is a graph showing the therapeutic effect of mFGF21 protein of the present invention on fatty liver in HFHC feed-induced NASH model mice;

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Description of the drawings: the procedures of designing, synthesizing and cloning the gene, constructing an expression vector, extracting nucleic acid, sequencing and identifying, and isolating and purifying the expression product, which are involved IN the present invention, can be performed according to the techniques known IN the art (see CURRENT promoters IN MOLECULAR BIOLOGY). Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1

Expression and purification of FGF21 protein

(1) Construction of expression vectors

The designed mFGF21 gene (shown in SEQ ID NO:1 of a sequence table) and the wild hFGF21 gene (GenBank: PA260867.1) are synthesized by Nanjing Kinshire company, and Nde I and Hind III enzyme cutting sites are added at both ends of a target gene. The synthesized target gene containing the cleavage site was ligated into pET30a (+) vector, and then transformed into E.coli DH 5. alpha. each. And (3) selecting positive clones, carrying out enzyme digestion identification and sequencing to obtain the successfully constructed recombinant expression vectors pET30a-mFGF21 and pET30a-hFGF 21.

(2) Expression identification of FGF21 protein

The recombinant expression vectors pET30a-mFGF21 and pET30a-hFGF21 were transformed into BL21(DE3) competent cells, respectively. Single colonies were picked and inoculated into 20mL LB medium containing Kan (50. mu.g/mL), respectively, and cultured at 37 ℃ for 12 hours, and inoculated into another 20mL LB medium containing Kan (50. mu.g/mL) at a volume ratio of 1:100, cultured at 37 ℃ and 180rpm, when A600 was around 0.5, IPTG (isopropyl thiogalactoside) was added to a final concentration of 0.5mmol/L, and further cultured at a rotation speed of 100rpm and induced at 37 ℃ for 4 hours, and then the cells were harvested. The cells were resuspended in PBS (phosphate buffered saline), disrupted, centrifuged, and the supernatant and pellet were separated and analyzed by 12% SDS-PAGE. The results show that mFGF21 and hFGF21 proteins are both expressed in E.coli in the form of inclusion bodies, and the expression level of mFGF21 protein is higher than that of hFGF21 protein (as shown in FIG. 1), and the expression level of mFGF21 protein is about 700mg/L, and the expression level of eukaryotic expression system, such as mammalian cell system, is about 50mg/L according to the literature reports (Chinese Proc. of biochemistry and molecular biology, 2012, 28 (8): 768-774); the Pichia expression system was about 30mg/L (Molecular Medicine Reports, 2016; 13: 3619-; the yield of prokaryotic systems reported in the literature is about 71mg/L (Appl Microbiol Biotechnol.2019; 103(2): 719-730.). The high-density fermentation system of the escherichia coli in the patent can achieve the yield of 400 mg/L. Therefore, it was demonstrated that a higher expression level can be obtained by using the E.coli system. Lane M: a protein standard molecular weight Marker; YQ: pre-induction thalli; and (3) QJ: performing whole bacteria after induction; SQ: crushing thallus and centrifuging supernatant; CD: and (4) crushing the thallus, centrifuging and precipitating.

(3) Purification of FGF21 protein

Collecting high-density fermentation expression thallus by a tubular centrifuge, washing the thallus by using a washing solution (20mmol/L Tris, 150mmol/L NaCl, pH8.0 buffer solution) for 3 times, adding lysozyme (1mg/mL) into the harvested thallus, incubating for 30min at 4 ℃, homogenizing at 1000bar under high pressure for 2 times to break the thallus. Continuously adopting a tubular centrifuge to enrich and wash the inclusion body (4 times of pH8.0 washing liquid is used for washing and filtering); after the inclusion body is dissolved by using the 6M urea-containing denatured liquid with the pH of 8.0, the renaturation protein is diluted by the 1M urea-containing renaturation liquid with the pH of 40 times of the pH of 8.0, and finally the renaturation liquid is concentrated and replaced by buffer solution through a 10kD aperture membrane or a hollow fiber column, so that the target protein with good renaturation is obtained.

And (3) purifying the renatured protein by using a two-step Q anion exchange chromatographic column, purifying the FGF21 protein by increasing the concentration of salt ions and eluting, and finally concentrating and replacing a buffer solution by using a 10kD pore membrane or hollow fiber column to obtain the target FGF21 protein.

The purity and content of FGF21 protein prepared by the expression and purification of the bacterial cells in example 1 were analyzed by SDS-PAGE electrophoresis and high performance liquid chromatography. The result shows that the electrophoresis and liquid phase purity of the purified variant mFGF21 protein is more than 98%, and the liquid phase purity of the wild type hFGF21 protein is about 97% (as shown in FIG. 2). Through computational analysis, the yield of the overall purification preparation process of the mFGF21 protein (about 400mg/L) is remarkably higher than that of the hFGF21 protein (about 80 mg/L).

Stability of the mFGF21 protein and hFGF21 protein prepared in example 1 was tested in vitro and in vivo. The specific methods and results are as follows:

filtering and sterilizing mFGF21 and hFGF21 by a 0.22-micron filter membrane, mixing the mixture with mouse plasma according to the final protein concentration of 0.05mg/ml, incubating the mixture at 37 ℃ for 12 hours, 24 hours, 36 hours and 48 hours respectively, sampling, and detecting the protein degradation degree by an ELISA double-antibody sandwich method. 89% of the reactivity of the mFGF21 protein was retained after 48h incubation with plasma, whereas the wild-type hFGF21 showed only about 21% of the reactivity (as shown in FIG. 3,^##P<0.01,^###P<0.001vs hFGF21), indicating that the variant mFGF21 protein has good plasma stability relative to the hFGF21 protein.

10 SD rats weighing about 300g were selected and randomly divided into 2 groups. Each group is injected with hFGF21 and mFGF21 subcutaneously with the dose of 1mg/kg, blood is collected for about 300 mu L at 0h, 5min, 15min, 30min, 45min, 1h, 2h, 4h, 8h, 12h and 24h after administration, centrifugation is carried out for 10min at 12000r/m, and the supernatant is taken for protein content detection. Determination of in vivo half-life of protein by ELISA double antibody sandwich method: the standard curves of protein concentration content are respectively established by using hFGF21 and mFGF21 proteins (2 mu g/mL, 0.2 mu g/mL, 200ng/mL, 20ng/mL and 2ng/mL) which are diluted with different concentrations, the content of the target protein in each serum is determined by ELISA, and the in vivo half-life of 2 proteins is statistically analyzed and calculated. Half life t in vivo_1/2＝0.301*(t₂-t₁)/log(OD₁/OD₂) Wherein OD₁And OD₂Respectively represent t₁And t₂The average light absorption value on the ELISA plate corresponding to the serum is taken out. The results are shown in FIG. 3(^##P<0.01vs hFGF21), the in vivo half-life of hFGF21 and mFGF21 is calculated by the formula to be about 36min and 155min respectively, which shows that the in vivo half-life of the modified variant mFGF21 protein is obviously improved relative to the wild type hFGF21 protein.

The mFGF21 protein and hFGF21 protein prepared in example 1 are tested for sugar absorption activity in vitro, and the specific methods and test results are as follows:

HepG2 cell culture: HepG2 cell is human liver cancer cell line, the culture condition is high-sugar DMEM culture medium, 10 wt% newborn bovine serum NCS, penicillin 100 mug/mL and streptomycin 100 mug/mL are added, and the volume fraction is 5% CO at 37 DEG C₂And culturing under saturated humidity condition. When the cells are grown to a high density, passage should be performed, after digestion with 0.25 wt% trypsin solution, the cells are inoculated in a new culture flask for culture in a volume ratio of 1:3 to 1:5, and the cells in the logarithmic growth phase are taken for experiment.

Inoculation and treatment of HepG2 cells: digesting HepG2 cells in good growth state with 0.25% trypsin digestion solution, centrifuging to collect cells, and performing 2.5X 10⁴The cells were seeded in a 96-well plate and cultured in a volume of 200. mu.L per well. When the cells grow to a uniform monolayer, the upper layer of culture solution is discarded, and fresh serum-free culture medium is added for further culture for 12 hours. Cells were stimulated with different concentrations (10, 100, 1000nmol/L) of hFGF21 and mFGF21 proteins for 24h, respectively. Taking the culture medium supernatant, detecting the glucose content in the culture medium by GOD-POD method, repeating the detection for at least 3 times per well, reacting at 37 deg.C for 5-10min, and measuring OD value at 500nm wavelength. The glucose consumption rate was calculated and the experimental results were analyzed using statistics.

The calculation formula of the glucose concentration in the culture solution and the cell glucose consumption rate is as follows:

glucose concentration (mmol/L) ═ OD_{Sample (I)}/OD_{Standard of merit}×5.55mmol/L

Cell glucose consumption rate (%) - (C)_{Blank glucose}-C_{Administration of glucose})/C_{Blank glucose}]×100％

The results are shown in FIG. 4 (^#P<0.05,^##P<0.01,^###P<0.001vs hFGF21), the absorption rate of two proteins for promoting cell sugar is dose-dependent, and the absorption rate of mFGF21 for stimulating cell sugar at three concentrations of low, medium and high is obviously higher than that of hFGF21 protein, which shows that the in vitro activity of variant mFGF21 protein is obviously better than that of hFGF21 protein.

The efficacy of the mFGF21 protein and hFGF21 protein prepared in example 1 in NASH model mice was evaluated by the following methods and results:

experimental animals and breeding: the C57BL/6 mouse and animal experiments were carried out by Jiangsu Jiejiaokang Biotech limited.

40 SPF-grade 6-week-old male C57BL/6 mice are taken, fed with high-fat high-cholesterol high-fructose feed (HFHC) for 4 months, the abnormal body weight is eliminated, 30 adult mice with relatively close-average blood sugar and body weight values are screened, and the adult mice are randomly divided into a model control group (Vehicle group), an hFGF21 group and an mFGF21 group, wherein each group comprises 10 mice. Another 10 same-week-old male C57BL/6 mice were used as a Normal control group (Normal group). The corresponding test substance is given to the experimental group for one time at 8 o' clock in the morning, and subcutaneous injection is carried out, and the dosage is 2 mg/kg; the Vehicle and Normal groups were injected with the same volume of saline for 4 consecutive weeks. During the experiment, the patient can eat and drink water freely. During which time the mice were monitored for diet and body weight. 3 days after administration, fasting blood glucose levels were measured in each experimental group of mice (6 h). After 4 weeks of administration, each experimental group of mice was sacrificed (fasting overnight), and serum glutamic-oxaloacetic transaminase (AST), glutamic-pyruvic transaminase (ALT), Triglyceride (TG), total Cholesterol (CHO), high-density lipoprotein cholesterol (HDL), and low-density lipoprotein cholesterol (LDL) levels of the experimental mice were measured and liver pathological tissue section staining and NASH index detection were performed. Statistical analysis was performed on all experimental data.

The fasting blood glucose level of 6h in each experimental group of mice before and after 3d administration is shown in FIG. 5: (^*P<0.05,^**P<0.01vs Vehicle；^#P<0.05,^##P<0.01vs hFGF21), the fasting blood glucose level of the mice of the mFGF21 group was significantly decreased relative to the model control group, and the effect of the mFGF21 protein on blood glucose improvement was also significantly better than that of the hFGF 21. The variant mFGF21 protein is proved to have good potential in treating diabetes.

The results of the body weight measurements of the mice in each experimental group are shown in FIG. 6: (^*P<0.05,^**P<0.01vs Vehicle；^#P<0.05,^##P<0.01 vs. hFGF21), compared with a model control group, mFGF21 and hFGF21 proteins can both obviously reduce the weight of the mice, while the capacity of the mFGF21 protein for controlling the weight of the mice is more obvious than that of the hFGF21 protein, and the weight of the mFGF21 mice rises slowly after drug withdrawal. The effect of the variant mFGF21 protein on weight loss is obviously better than that of hFGF 21.

The results of measurement of blood lipid levels and liver function parameters in the sera of mice of each experimental group 6 weeks after administration are shown in FIGS. 7 and 8: (^*P<0.05,^**P<0.01vs Vehicle；^#P<0.05,^##P<0.01vs hFGF21), the serum TG, CHO, LDL and AST and ALT levels in mice of two FGF21 protein groups are obviously reduced, and the HDL level is obviously increased compared with the model control group. Compared with hFGF21, the mFGF21 protein has more remarkable effect on improving the blood fat and liver function level of a model mouse. It is proved that the mFGF21 protein has better effect on treating hyperlipidemia and non-alcoholic fatty liver disease.

Fatty liver improvement, the experimental end points were HE-stained and oil red-stained liver tissue sections of mice of each experimental group, and the tissue sections were scored (scoring from three aspects of steatosis, inflammatory lesions, and ballooning). The judgment standard is as follows: the sum is less than 3 points, namely non-NASH; the total score is more than 5 points, namely NASH is obtained; and 3 points < the total score < 5 points, namely the nondeterministic NASH. Pathological section scoring results show that (see fig. 9, Bar is 50 μm), compared with a model control group, the hFGF21 and the mFGF21 protein have obvious effects on improving fatty liver of a model mouse, and according to the NASH score, the mFGF21 protein has a better treatment effect on fatty liver lesion improvement.

The performance test and the results of example 1 show that the invention designs and verifies a variant FGF21 protein with good stability and high biological activity, and simultaneously develops a high-efficiency purification method of FGF21 protein. Through a model animal efficacy evaluation test, the improvement and treatment effects of the variant FGF21 protein on glycolipid disturbance and fatty liver symptoms of NASH diseases are obviously better than those of wild type hFGF21 protein. In conclusion, the variant FGF21 protein and the derivatives thereof have good potential and application prospect in the aspect of treating metabolic diseases.

The foregoing embodiments illustrate the principles, principal features and advantages of the present invention, and those skilled in the art will understand that the present invention is not limited to the foregoing embodiments, which are merely illustrative of the principles of the present invention and are not intended to limit the invention. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention. The present invention is subject to various changes and modifications without departing from the scope of the present invention, and such changes and modifications are intended to be included within the scope of the present invention.

SEQUENCE LISTING

<110> innovative center of Jiangxiang Chinese medicine

<120> recombinant variant FGF21 protein and preparation method and application thereof

<130> innovative center of Jiangxiang Chinese medicine

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 543

<212> DNA

<213> SEQ ID NO:1

<400> 1

gctcctatac cagatagttc acccctatta caatttggtg gtcaagtgcg tcagcgctat 60

ctgtacaccg atgatgcaca gcaaaccgaa gcgcatctgg aaattcgtga ggacggcacg 120

gtgggtggtg cggcggatca gtctccggag agcctgctgc aactgcgtgc actggcgccg 180

ggcgtcatcc agattctggg tgttcatacc tcgcgcttct tatgccaacg cccagacggc 240

gctctgtacg gctccttgca cttcgacccg gaggcatgta gctttcgtga actgctgctt 300

gaggacggtt ataatgttta tcagagcgaa gcgcacggct tgccgttgca cttgccgggc 360

aaccgctccc cgcatcgtga tccggctccg cgtggtccgg cgagattcct gccgctgcct 420

ggtctgccgc cggcgctgcc ggagccgccg ggcatcctcg ccccacagcc gcccgacgtg 480

ggcagctccg atccgctcag catggttggt gccagccaag gtcgtagccc aagctacgaa 540

tct 543

<210> 2

<211> 181

<212> PRT

<213> SEQ ID NO:2

<400> 2

Ala Pro Ile Pro Asp Ser Ser Pro Leu Leu Gln Phe Gly Gly Gln Val

1 5 10 15

Arg Gln Arg Tyr Leu Tyr Thr Asp Asp Ala Gln Gln Thr Glu Ala His

20 25 30

Leu Glu Ile Arg Glu Asp Gly Thr Val Gly Gly Ala Ala Asp Gln Ser

35 40 45

Pro Glu Ser Leu Leu Gln Leu Arg Ala Leu Ala Pro Gly Val Ile Gln

50 55 60

Ile Leu Gly Val His Thr Ser Arg Phe Leu Cys Gln Arg Pro Asp Gly

65 70 75 80

Ala Leu Tyr Gly Ser Leu His Phe Asp Pro Glu Ala Cys Ser Phe Arg

85 90 95

Glu Leu Leu Leu Glu Asp Gly Tyr Asn Val Tyr Gln Ser Glu Ala His

100 105 110

Gly Leu Pro Leu His Leu Pro Gly Asn Arg Ser Pro His Arg Asp Pro

115 120 125

Ala Pro Arg Gly Pro Ala Arg Phe Leu Pro Leu Pro Gly Leu Pro Pro

130 135 140

Ala Leu Pro Glu Pro Pro Gly Ile Leu Ala Pro Gln Pro Pro Asp Val

145 150 155 160

Gly Ser Ser Asp Pro Leu Ser Met Val Gly Ala Ser Gln Gly Arg Ser

165 170 175

Pro Ser Tyr Glu Ser

180

Claims

1. A recombinant variant mFGF21 protein having the amino acid sequence: shown as SEQ ID NO. 2.

2. The gene encoding the variant mFGF21 protein of claim 1, wherein the nucleotide sequence of the gene is represented by SEQ ID NO. 1 of the sequence Listing.

3. A vector or host cell carrying the gene encoding according to claim 2.

4. A method of producing the recombinant variant mFGF21 protein of claim 1, comprising the steps of:

the method comprises the following steps: connecting the synthesized gene segment of SEQ ID NO. 1 in the sequence table with a prokaryotic expression vector, and transforming escherichia coli to obtain an expression strain;

step two: culturing the recombinant expression strain and inducing the recombinant expression strain to produce the target protein;

step three: and (4) harvesting the expression strain treated in the step two, and performing separation and purification procedures on the expression strain to obtain the target protein.

5. A pharmaceutical composition comprising the mFGF21 protein of claim 1 or the mFGF21 protein prepared by the preparation method of claim 4.

6. A pharmaceutical composition according to claim 5, further comprising a pharmaceutically acceptable carrier, excipient or diluent.

7. Use of a pharmaceutical composition according to claim 5 or 6 for the preparation of a medicament for the prevention or treatment of metabolic disorders including diabetes, obesity, hyperlipidemia and non-alcoholic fatty liver disease.