WO2018196616A1 - 人fgf21突变体、其制备方法及用途 - Google Patents

人fgf21突变体、其制备方法及用途 Download PDF

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WO2018196616A1
WO2018196616A1 PCT/CN2018/082669 CN2018082669W WO2018196616A1 WO 2018196616 A1 WO2018196616 A1 WO 2018196616A1 CN 2018082669 W CN2018082669 W CN 2018082669W WO 2018196616 A1 WO2018196616 A1 WO 2018196616A1
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amino acid
fgf21
mutant
acid sequence
protein
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French (fr)
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王俊峰
赵宏鑫
朱磊
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中国科学院合肥物质科学研究院
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Priority to US16/608,706 priority Critical patent/US11746134B2/en
Priority to JP2020509145A priority patent/JP7097434B2/ja
Priority to EP18791988.1A priority patent/EP3636663A4/en
Publication of WO2018196616A1 publication Critical patent/WO2018196616A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/50—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • the invention belongs to the field of protein engineering, in particular to a human fibroblast growth factor 21 (FGF21) mutant, a gene encoding the same, and a preparation method of the mutant and the use of the mutant for treating type 2 diabetes, obesity and abnormality A method of dyslipidemia or metabolic disorders.
  • FGF21 human fibroblast growth factor 21
  • FGF21 belongs to the secreted protein of the fibroblast growth factor (FGF) 19 subfamily, which includes FGF19, FGF21, and FGF23 (Itoh et al., 2004, Trend Genet. 20: 563-69). ). FGF21 is an atypical FGF that is independent of heparin and functions in glucose, lipids and energy metabolism. It promotes glucose uptake in adipocytes by upregulating GLUT1 expression, the mechanism of which is different from insulin. In rodents, monkeys and humans with diabetes, FGF21 lowers the fasting serum concentration of glucose and lowers the fasting serum concentrations of triglycerides, insulin and glucagon.
  • FGF fibroblast growth factor
  • FGF21 farnesoid growth factor21
  • mice Furthermore, administration of FGF21 resulted in a dose-dependent loss of cumulative body weight in a diet-induced obese rodent model.
  • Experimental studies have provided support for the pharmacological administration of FGF21 for the treatment of diabetes, obesity, dyslipidemia, and metabolic syndrome.
  • Human FGF21 is easily degraded, used in a large amount, and has poor stability and efficacy. Therefore, it is necessary to modify wild type FGF21 to improve the effectiveness and stability of human FGF21 to reduce the dose administered to patients.
  • the present invention provides an alternative human FGF21 mutant, a coding gene thereof, and a preparation method and use of the mutant.
  • the invention provides a human FGF21 mutant having the following amino acid changes based on the amino acid sequence of the wild-type human FGF21 protein:
  • the 171th Pro mutation of the amino acid sequence of the wild type human FGF21 protein is Gly; and/or
  • amino acid fragment at positions 24 to 31 of the amino acid sequence of the wild type human FGF21 protein is 5-15 amino acids, preferably 6-14 amino acids, more preferably 7-13 amino acids, still preferably 8-10 amino acids, most A fragment replacement of 8 amino acids is preferred.
  • the amino acid fragment for the substitution is a combination of any of the amino acids.
  • the amino acid fragment for the substitution is a fragment of 8 amino acids, represented by the formula X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 .
  • X 1 -X 8 are each independently selected from any amino acid, preferably X 1 is Ser or Asp, X 2 is Gly or Asp, X 3 is Pro or Ala, X 4 is Ala or Gln, X 5 is Gly or Gln, X 6 is Leu or Tyr, X 7 is Ser or His, and X 8 is Ser or Ala.
  • the present inventors based on the spatial structure of FGF21, the present inventors have found that the amino acid P at position 171 of the CGF end of FGF21 is easily exposed to the surface and is easily degraded by the enzyme, and the present invention mutates it to G, thereby increasing the stability of FGF21. However, it did not affect the activity of FGF21 (data not shown). Further, based on the structure, the present inventors found that the space structure of the N-terminal 24-31 of FGF21 is a loop loop, and the length of the loop loop retains the activity of FGF21 while having a significant influence on the stability of FGF21.
  • the inventors have also found that the 31st and 32nd positions of wild-type FGF21 are spatially close to the 43th and 44th amino acids, but the structure is not sufficiently stable.
  • the inventors have unexpectedly discovered that, without affecting the length of the loop loop between positions 23-32, after inserting a cysteine Cys between the 31st and the 32nd, the half Cystine stabilizes its structure by forming a disulfide bond with the cysteine mutated at position 43 or 44; in addition, the amino acid on the loop loop is mutated, and the Cys inserted between the 31st and 32nd positions
  • the 43th or the 44th mutated Cys forms a disulfide bond and stabilizes the structure, and changes the amino acid composition of the 24-32 Loop loop to sufficiently increase the activity of FGF21.
  • the laboratory has solved the structure of FGF21.
  • the CD experiment and NMR experiments fully demonstrate that the mutant forms a more stable structure after forming a disulfide bond.
  • FGF21 was used to change the amino acid composition of the loop loop before and after the change, and the activity after the change was significantly higher than that before the change.
  • the amino acid sequence of the wild-type human FGF21 protein forms a stable disulfide bond between the Cys increased at positions 31-32 and the Cys at position 43 or 44, and is between positions 23-32 of wild FGF21.
  • the length of the amino acid has an obvious effect on the stability of FGF21. Therefore, based on the disclosure of the present specification, those skilled in the art can arbitrarily select the length of the fragment and the amino acid species to make desired changes or improvements in the effects of the present invention.
  • the amino acid fragment for said substitution is DDAQQTEA (the corresponding mutant is also referred to as FGF21-AG hereinafter), and the nucleic acid sequence encoding the mutant is shown in SEQ ID NO: 3, amino acid The sequence is shown in SEQ ID NO:4.
  • the amino acid fragment for the substitution is SGPHGLSS (the corresponding mutant is also referred to hereinafter as FGF21-LG), and the nucleic acid sequence encoding the mutant is set forth in SEQ ID NO: 5.
  • the amino acid sequence is shown in SEQ ID NO: 6.
  • wild-type FGF21 is obtained by searching FGF21 from http://www.uniprot.org/, and selecting an organism (Homo sapiens) from the obtained results.
  • the human FGF21 is numbered (Q9NSA1) in uniprot, and we obtained 209 residues in the protein sequence of FGF21.
  • the N-terminal 28 amino acid signal peptide was removed, and the obtained FGF21 sequence has 181 residues, which is the wild type FGF21 sequence, the nucleic acid sequence is SEQ ID NO: 2, and the corresponding amino acid sequence is SEQ ID NO: 1.
  • wild type human FGF21 protein refers to a mature FGF21 protein (hereinafter also referred to as FGF21) naturally present in humans, and in a typical case, its amino acid sequence is SEQ ID NO: 1.
  • FGF21 mature FGF21 protein
  • SEQ ID NO: 1 An amino acid sequence having 90% or more, 95% or more, preferably 98% or more, more preferably 99% or more homology with SEQ ID NO: 1.
  • % homology relative to a reference polypeptide sequence is defined as the alignment of the sequences and the introduction of gaps as necessary to achieve maximum percent sequence identity without considering any conservative substitutions as sequence identity.
  • BLAST BLAST-2
  • ALIGN ALIGN
  • Megalign DNASTAR
  • % homology values were generated using the sequence comparison computer program ALIGN-2.
  • the author of the ALIGN-2 sequence comparison computer program is Genentech, Inc., and the source code has been submitted with the user documentation to the U.S. Copyright Office, Washington D.C., 20559, which is registered under U.S. Copyright Registration No. TXU510087.
  • the ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or the program can be compiled from source code.
  • the ALIGN-2 program should be compiled for use with UNIX operating systems, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not need to be changed.
  • the invention provides a nucleic acid encoding any of the mutants described above.
  • the invention also provides a vector comprising the nucleic acid described above.
  • the vector is an expression vector, more preferably a prokaryotic expression vector.
  • the invention also provides a host cell comprising the vector described above.
  • the host cell is an E. coli cell.
  • the present invention also provides a method for producing a human FGF21 mutant, the method comprising operably linking the coding gene of the human FGF21 mutant to the expression vector to obtain a recombinant expression vector; and transforming the recombinant expression vector into a host Cells; cultured recombinant host cells, induction of recombinant protein expression, collection and purification of expressed proteins.
  • the method comprises sequentially ligating a SUMO tag gene (6 hiss tag at the N-terminus of the SUMO tag gene) and a nucleic acid sequence encoding the human FGF21 mutant.
  • the gene is operably linked to the expression vector to obtain a recombinant expression vector; the recombinant expression vector is transformed into a host cell, the recombinant host cell is cultured, the expression of the recombinant protein is induced, the expressed protein is collected and purified, and the SUMO enzyme is cleaved and purified.
  • the host cell is an E. coli cell.
  • the expression condition is: 10-37 ° C cultured cells until the OD 600 reaches 0.2-1.0, the final concentration of 0.1-1 mM IPTG is added, and the expression is induced at 4-37 ° C. -20h.
  • the cells are cultured at 37 ° C to an initial concentration of 1 mM IPTG at an OD 600 of 0.8 and induced for 6 h at 25 °C.
  • the collected cells are resuspended in a lysis buffer (20Mm Tris-HCl 100Mm NaCl, pH 8.0), the cells are disrupted, centrifuged, and the supernatant is collected, and the supernatant is mixed with the purified filler.
  • a lysis buffer (20Mm Tris-HCl 100Mm NaCl, pH 8.0)
  • the mixture is transferred to a chromatography column for purification of the protein, and the purified SUMO-fused human FGF21 mutant protein is digested with SUMO enzyme, and the conditions include: temperature: 0-37 ° C
  • the buffer is 20Mm Tris-HCl 100mM NaCl pH8.0, PBS, Tris-HCl, pH 6.8-8.0, cleavage 0-24 hours, using Ni metal chelate affinity chromatography to obtain human FGF21 mutant, and then The FGF21 mutant protein was repurified by size exclusion chromatography.
  • the invention provides the use of the mutant in the manufacture of a medicament for the treatment of type 2 diabetes, obesity, dyslipidemia, or metabolic syndrome.
  • the invention provides a pharmaceutical composition comprising a mutant of the invention and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier refers to carriers and auxiliary substances such as diluents or excipients which are compatible with the other ingredients of the formulation.
  • pharmaceutical composition includes a product comprising a predetermined amount or ratio of a particular ingredient, as well as any product obtained directly or indirectly by combining a particular amount of a particular ingredient.
  • it comprises a product comprising one or more active ingredients, and optionally a carrier comprising an inert ingredient, and a combination, combination or aggregation of any two or more ingredients, or one or more ingredients Any product that decomposes, or is obtained directly or indirectly from other types of reactions or interactions of one or more constituents.
  • “Mutation” as used in the present invention includes substitution, deletion, and addition of an amino acid.
  • the mutant of the present invention has the advantage that wild type FGF21 does not have. Such advantages include improved pharmacological efficacy and/or improved drug stability.
  • the FGF21 mutant protein of the invention has one or more advantageous physiological characteristics, including a more pronounced pharmacological effect in vivo, with greater thermostability. Further, the FGF21 variant of the present invention has a potential effect on type 2 diabetes, obesity, dyslipidemia, or metabolic syndrome, or any treatment thereof.
  • Figure 1 Schematic representation of the SUMO-FGF21 mutant expression vector.
  • Electrophoresis pattern of purified SUMO-FGF21-LG nucleic acid M is DL5000 DNA Marker, and 1 is a purified product of SUMO-FGF21-LG gene.
  • FIG. 1 Electropherogram of SUMO-FGF21-AG nucleic acid after purification, M is DL5000 DNA Marker, and 1 is a purified product of SUMO-FGF21-AG gene.
  • the main reagents and instruments used in the experiment were: the primers used in the cloning were all synthesized by Shanghai. Polymerase chain reaction (PCR) DNA polymerase (primer star), restriction endonuclease, ligase, Dpn I were purchased from Takara; DNA marker was purchased from Thermo; common DNA product recovery kit, agarose The gel recovery kit was purchased from TIANGEN. E. coli DH5 ⁇ strain, E. coli BL21 strain, pET22b-SUMO-FGF21 plasmid, SUMO protease purchased Beijing Suo Laibao Technology Co., Ltd. Tris, imidazole was purchased from Shanghai, other salts were purchased from Sinopharm, and concentrated tubes were purchased from Millipore. FPLC instruments use GE's The system, the Ni-Sepharose column and the molecular exclusion chromatography column Hiload 16/60 Superdex 75pg were purchased from GE. The BCA kit was purchased from Thermo.
  • a 1-8 position is a protected base, and positions 9 to 14 are restriction endonuclease sites;
  • the 1-3th position of b is a protected base, and the 4th to 9th positions are restriction endonuclease sites.
  • the first mutation was carried out using the pET22b-SUMO-FGF21 plasmid as a template: PCR site-directed mutagenesis was carried out using the upstream primer 171G-F and the downstream primer 171G-R. A FGF21-P171G mutant was obtained. The second mutation was carried out by using the FGF21-P171G mutant as a template, 43C-F as the upstream primer and 43C-R as the downstream primer for PCR site-directed mutagenesis to obtain the FGF21-G43C-P171G mutant.
  • the third mutation using the FGF21-G43C-P171G mutant as a template, 31-32C-F as the upstream primer and 31-32C-R as the downstream primer for PCR site-directed mutagenesis, the FGF21-AG mutant was obtained.
  • the fourth mutation using FGF21-AG as a template, FGF21-LG-F as the upstream primer, and FGF21-LG-R as the downstream primer for the mutation.
  • a FGF21-LG mutant was obtained.
  • the PCR reaction conditions are as follows.
  • the PCR reaction system and reaction procedure of the above mutation process are as follows:
  • PCR reaction system 2 ⁇ Prime STAR HS (Premix) 25 ⁇ l, upstream primer (10 ⁇ M) 1 ⁇ l, downstream primer (10 ⁇ M) 1 ⁇ l, template 1 ⁇ l, ddH 2 O 22 ⁇ l, total volume 50 ⁇ l.
  • step 1 pre-denaturation temperature 98 ° C 10 mins; second step, denaturation temperature 98 ° C 10 secs; 3, annealing temperature 57 ° C 5 secs; step 4, extension temperature 72 ° C, 1 min. Repeat 30 cycles from steps 2 through 4.
  • the product after each mutation was digested with Dpn I enzyme for 1 h at 37 °C.
  • the digested product was subjected to agarose gel electrophoresis, and the plasmid was recovered by a gel recovery kit.
  • 1 ul of the plasmid was placed in 100 ul of competent cells, and the cells were placed on ice for 30 minutes, heat shocked at 42 ° C for 90 seconds, and 400 ul was added.
  • the LB medium was cultured at 37 ° C for 45 minutes with shaking, then plated, and inverted at 37 ° C overnight.
  • the monoclonal antibody was used as a template, and SUMO-F was used as the upstream primer and FGF21-R as the downstream primer for PCR identification.
  • the identification results of FGF21-LG are shown in Fig. 2, and the identification structure of FGF21-AG is shown in Fig. 3.
  • the successfully identified FGF21 mutant was subjected to gene sequencing and results analysis.
  • the samples were sequenced by Bioengineering (Shanghai) Co., Ltd. Sequence analysis was performed using DNAMAN software.
  • the result of the sequence determination the nucleic acid sequence of the mutant FGF21-AG is shown in SEQ ID NO. 3, the corresponding amino acid sequence is shown in SEQ ID NO. 4; the nucleic acid sequence of the mutant FGF21-LG is shown in SEQ ID NO.
  • the corresponding amino acid sequence is shown in SEQ ID NO.
  • a schematic diagram of its construction carrier is shown in Fig. 1.
  • the successfully sequenced plasmid containing the nucleic acid sequence encoding the FGF21 mutant was transformed into the BL21 (DE3) strain, and the monoclonal strain was picked for expansion culture, and the strain was stored in a -80 ° C refrigerator.
  • the FGF21 mutant strain was inoculated into 5 ml of LB medium (containing 100 ⁇ g/ml ampicillin) at a seeding rate of 2%, and cultured overnight at 37 ° C, shaking at 220 rpm, and the overnight culture broth was inoculated into each bottle at 2%, each A 500 ml triangular bottle of 500 ml LB medium (containing 100 ⁇ g/ml ampicillin). Grown to OD 600 was added 1.0M IPTG 500ul of 0.8, induction of the expression of four hours at 25 °C. After centrifugation at 4 ° C, 8000 r / min for 10 minutes, the supernatant was discarded, and the cells were collected, and the cells were frozen at -80 ° C.
  • the expressed cells were resuspended in lysis buffer (50 mM Tris, 300 mM NaCl, pH 8.5), and then subjected to sonication (30% power, 2 seconds, 5 seconds, total time 45 minutes), 14000 rpm at 4 °C Centrifuge for 40 minutes and collect the supernatant.
  • the supernatant was added to a Ni-Sepharose column which had been pre-equilibrated with buffer, and mixed by rotation at 4 ° C for 30 minutes. After the solution was passed out, it was washed with 5 column volumes of buffer to remove unbound protein and impurities, and then 5 times column volume of buffer containing 30 mM imidazole (50 mM Tris, 300 mM NaCl, pH 8.5).
  • the heterologous heteroprotein was eluted, and the target protein was eluted with 5 column volumes of buffer containing 300 mM imidazole (50 mM Tris, 300 mM NaCl, pH 8.5), and the purification result was detected by SDS-PAGE (the results are shown in the figure). 4).
  • the dialysis fusion protein was detected by adding SUMO enzyme at a molecular molar ratio of 0.1%, and digesting at 4 ° C for 2 h, adding a final concentration of 20 mM imidazole to the digested protein solution, and then adding to the already used buffer ( On a pre-equilibrated Ni-Sepharose column of 50 mM Tris, 300 mM NaCl, pH 8.5, the mixture was spun at 4 ° C for 30 minutes.
  • the flow-through solution was collected, and the sample was concentrated to 10 mg/ml with a concentrating tube, and then purified by Superdex 75 molecular exclusion chromatography column, and subjected to molecular exclusion chromatography buffer (20 mM PBS, 100 mM NaCl, pH 7.2).
  • the protein is a FGF21 mutant protein, and FGF21 and mutants such as FGF21-LG and FGF21-AG proteins in the experiment are all purified by the above method.
  • the results of SDS-PAGE gel electrophoresis of FGF21-LG protein are shown in Figure 5
  • the results of SDS-PAGE gel electrophoresis of FGF21-AG protein are shown in Figure 6.
  • mice purchased in the Animal Model Institute of Nanjing University
  • the ob/ob mice purchased in the Animal Model Institute of Nanjing University
  • the implementation method was subcutaneous injection in the back. 0.1 mg/kg/day, continuous administration for seven days, and the blood glucose level before daily administration was recorded.
  • the FGF21-LG (Fig. 7) group showed stronger than the wild type FGF21 protein.
  • the efficacy of the drug was carried out with FGF21-AG, the embodiment was 0.6 mg/kg/day, and the blood glucose level before daily administration was recorded. The experiment shows that FGF21-AG also has obvious hypoglycemic effect, and the results are shown in Fig. 8.
  • the body weight loss test of the animals was carried out using the FGF21 mutant protein.
  • the ob/ob mice were divided into 4 groups, namely, vehicle control group, ie, normal saline, FGF21 group and human FGF21-LG group, and FGF21-AG group, 8 rats in each group.
  • the purified protein was administered at 0.6 mg/kg/day for 6 consecutive days, and the body weight change was recorded after 6 days.
  • the wild-type FGF21 protein has a more pronounced weight-reducing effect and is more potent.
  • the mutant according to the invention has a greater thermal stability than the wild type.

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Abstract

提供了人成纤维细胞生长因子21(FGF21)突变体及其编码基因，制备所述突变体的方法，和治疗2型糖尿病、肥胖、异常血脂症、或代谢紊乱的方法。

Description

人FGF21突变体、其制备方法及用途 技术领域
本发明属于蛋白质工程领域，具体涉及人成纤维细胞生长因子21(FGF21)突变体，其编码基因，以及所述突变体的制备方法和使用所述突变体用于治疗2型糖尿病、肥胖、异常血脂症、或代谢紊乱的方法。
背景技术
FGF21属于成纤维细胞生长因子(FGF)19亚家族的分泌蛋白，所述成纤维细胞生长因子(FGF)亚家族包括FGF19、FGF21、和FGF23(Itoh等，2004，Trend Genet.20:563—69)。FGF21是一种非典型的FGF，其独立于肝素，并在葡萄糖、脂类和能量代谢中发挥功能。其通过上调GLUT1表达，促进脂肪细胞中的葡萄糖摄取，其机制不同于胰岛素。在患有糖尿病的啮齿动物、猴子和人中，FGF21降低葡萄糖空腹血清浓度，并降低甘油三酯、胰岛素和胰高血糖素的空腹血清浓度。此外，在饮食诱导的肥胖啮齿类动物模型中，施用FGF21导致累计体重呈剂量依赖性丧失。实验研究提供了FGF21具有用于治疗糖尿病、肥胖、异常血脂症、代谢综合症的药物学给药的支持。
人FGF21易降解，用量大，稳定性及药效较差，因此需要对野生型FGF21进行改造，提高人FGF21的有效性和稳定性，以减少对患者给药的剂量。
发明概述
为了解决上述问题，本发明提供了替代性的人FGF21突变体，其编码基因以及所述突变体的制备方法和用途。
具体而言，本发明一方面提供了一种人FGF21突变体，其在野生型人FGF21蛋白氨基酸序列的基础上具有以下氨基酸改变：
1)在野生型人FGF21蛋白氨基酸序列的第31位Ala和第32位His之间增加一个Cys，并且第43位Gly、第44位Ala中的任意1个或2个位置处的氨基酸突变为Cys，其中这两个Cys形成分子内二硫键，
2)野生型人FGF21蛋白氨基酸序列的第171位Pro突变为Gly；和/或
3)野生型人FGF21蛋白氨基酸序列的第24位至31位的氨基酸片段用5-15个氨基酸，优选6-14个氨基酸，更优选7-13个氨基酸，还优选8-10个氨基酸，最优选8个氨基酸的片段替换。
在优选的实施方案中，用于所述替换的氨基酸片段是任意氨基酸的组合。
本文中使用的氨基酸的缩写如下：
氨基酸 3-字母 1-字母
丙氨酸 Ala A
精氨酸 Arg R
天冬酰胺 Asn N
天冬氨酸 Asp D
半胱氨酸 Cys C
谷氨酸 Glu E
谷氨酰胺 Gln Q
甘氨酸 Gly G
组氨酸 His H
异亮氨酸 Ile I
亮氨酸 Leu L
赖氨酸 Lys K
甲硫氨酸 Met M
苯丙氨酸 Phe F
脯氨酸 Pro P
丝氨酸 Ser S
苏氨酸 Thr T
色氨酸 Trp W
酪氨酸 Tyr Y
缬氨酸 Val V
在优选的实施方案中，用于所述替换的氨基酸片段为8个氨基酸的片段，用式X 1X 2X 3X 4X 5X 6X 7X 8表示。
在优选的实施方案中，X 1-X 8各自独立地选自任意氨基酸，优选X 1是Ser或Asp，X 2是Gly或Asp，X 3是Pro或Ala，X 4是Ala或Gln，X 5是Gly或Gln，X 6是Leu或Tyr，X 7是Ser或His，X 8是Ser或Ala。
本发明的方案中，基于FGF21的空间结构，本发明人发现，FGF21C端的第171位的氨基酸P容易暴露在表面易被酶所降解，本发明将其突变为G，从而 增加了FGF21稳定性，但不影响FGF21的活性(数据未显示)。进一步地，本发明人基于结构发现，FGF21的N端的第24-31的空间结构为一段loop环，loop环的长度在对FGF21的稳定性有显著影响的情况下仍然保持FGF21的活性。此外，基于FGF21的空间结构，本发明人还发现野生型FGF21的第31位和第32位与第43位和第44位的氨基酸在空间上是靠近的，但是结构不够稳定。经过深入研究，本发明人出人意料地发现：在不影响第23-32位间loop环的长度的情况下，在第31位和第32位之间插入了一个半胱氨酸Cys后，该半胱氨酸通过与第43位或第44位突变的半胱氨酸形成二硫键，从而稳定了其结构；另外，突变loop环上的氨基酸，在插入在第31-32位间的Cys与第43位或与第44位突变的Cys形成二硫键并稳定结构的基础上改变第24-31位Loop环的氨基酸组成，能够充分提高FGF21的活性。本实验室已经解析出FGF21的结构。通过CD实验和NMR实验充分说明了突变体形成二硫键后更加稳定了结构。本实验室做了FGF21改变loop环的氨基酸组成前后的对比，改变后的活性明显高于改变前的活性。
在本发明的方案中，野生型人FGF21蛋白氨基酸序列在31-32位增加的Cys和第43位或44位的Cys之间形成稳定二硫键时，在野生FGF21第23-32位间的氨基酸的长度对于FGF21的稳定性影响效果明显。因此，在本说明书公开内容的基础上，本领域技术人员能够任意选择所述片段的长度和氨基酸种类来对本发明进行效果可以预期的改变或改进。
在优选的实施方案中，用于所述替代的氨基酸片段是DDAQQTEA(其相应的突变体在下文中也称为FGF21-AG)，编码该突变体的核酸序列如SEQ ID NO：3所示，氨基酸序列如SEQ ID NO：4所示。
在另一个优选的实施方案中，用于所述替代的氨基酸片段是SGPHGLSS(其相应的突变体在下文中也称为FGF21-LG)，编码该突变体的核酸序列如SEQ ID NO：5所示，氨基酸序列如SEQ ID NO：6所示。
本发明中“野生型FGF21”是从http://www.uniprot.org/中搜索FGF21，在得到的结果中选择物种(organism)为人类(Homo sapiens)而得到的。人源FGF21在uniprot的编号为(Q9NSA1)，我们得到FGF21的蛋白质序列有209个残基。去除N端28个氨基酸的信号肽，得到的FGF21的序列有181个残基，即为野生型FGF21序列，其核酸序列为SEQ ID NO:2，相应氨基酸序列为SEQ ID NO:1。
因此，本发明中所述的“野生型人FGF21蛋白”是指天然存在于人中的成熟FGF21蛋白(下文中也称为FGF21)，在典型的情况下，其氨基酸序列如SEQ ID NO：1所示，或与SEQ ID NO：1具有90％以上，95％以上，优选98％以上，更优选99％以上的同源性的氨基酸序列。
相对于参比多肽序列的"％同源性"被定义为在对序列进行比对并且在必要时引入空隙(gap)以实现最大百分比序列同一性，而不将任何保守置换认为是序列同一性的一部分后，候选序列中与参比多肽序列中的氨基酸残基相同的氨基酸残基的百分比。为了确定百分比氨基酸序列同一性的比对可以以在本领域中的技术内的多种方式实现，例如，使用公众可获得的计算机软件如BLAST，BLAST-2，ALIGN或Megalign(DNASTAR)软件。本领域技术人员可以确定用于比对序列的合适参数，包括在比较的序列的全长上实现最大比对所需的任何算法。然而，对于本文来说，％同源性值使用序列比较计算机程序ALIGN-2产生。ALIGN-2序列比较计算机程序的作者是Genentech,Inc.，并且源代码已经与用户文件一起被提交于美国版权局,Washington D.C.,20559，其以美国版权登记号TXU510087被登记。公众可自Genentech,Inc.,South San Francisco,California获得ALIGN-2程序，或者所述程序可自源代码编译。ALIGN-2程序应当被编译为用于UNIX操作系统，包括数字UNIX V4.0D。所有序列比较参数由ALIGN-2程序设定并且不需要改变。
另一方面，本发明提供一种核酸，其编码以上所述的任一突变体。
本发明还提供一种载体，其包含以上所述的核酸。在优选的实施方案中，所述载体为表达载体，更优选为原核表达载体。
本发明还提供一种宿主细胞，其包含以上所述的载体。
在优选的实施方案中，所述宿主细胞为大肠杆菌(E.coli)细胞。
本发明还提供一种制备人FGF21突变体的方法，所述方法包括将所述人FGF21突变体的编码基因与所述表达载体可操作性的连接，得到重组表达载体；将重组表达载体转化宿主细胞；培养重组宿主细胞，诱导重组蛋白表达，收集并纯化表达的蛋白。
在优选的实施方案中，所述方法包括将SUMO标签基因(SUMO标签基因N端有6个his标签)和编码所述人FGF21突变体的核酸序列依次连接在一起。将该基因与表达载体可操作性的相连接，得到重组表达载体；将重组表达载体转 化宿主细胞，培养重组宿主细胞，诱导重组蛋白表达，收集并纯化所表达的蛋白，SUMO酶切割，再纯化。
所述的宿主细胞为大肠杆菌(E.coli)细胞。
本发明人发现，对于本发明所述的突变体，诱导表达条件为：10-37℃培养细胞到OD 600达到0.2-1.0时加入终浓度为0.1-1mM的IPTG，4-37℃诱导表达2-20h。在更优选的实施方案中，条件为37℃培养细胞到OD 600达到0.8时加入终浓度为1mM的IPTG，25℃诱导表达6h。
本发明的纯化方法是将收集到的菌体用裂解缓冲液(20Mm Tris-HCl 100Mm NaCl，pH 8.0)重悬，进行细胞破碎，离心，收集上清液，将上清液与纯化填料混合后孵育0.1-10个小时，将混合物转移到层析柱中，进行蛋白的纯化，将纯化得到的SUMO融合的人FGF21突变体蛋白用SUMO酶酶切，酶切条件包括：温度为0-37℃，缓冲液为20Mm Tris-HCl 100mM NaCl pH8.0，PBS，Tris-HCl，pH值为6.8-8.0，切割0-24小时，利用Ni金属螯合亲和层析，得到人FGF21突变体，再利用分子排阻层析对FGF21突变体蛋白进行再纯化。
另一方面，本发明提供所述突变体在制备药物中的用途，所述药物用于治疗2型糖尿病、肥胖、异常血脂症、或代谢综合征。
另一方面，本发明提供药物组合物，其包含本发明所述的突变体以及药用载体。术语“药用载体”是指与制剂的其他成分相容的载体和辅助物质如稀释剂或赋形剂。术语“药物组合物”包括包含预定量或比例的特定成分的产品，以及通过组合特定量的特定成分直接地或间接地得到的任何产品。优选地，它包括包含一种或多种活性成分，和任选的包含惰性成分的载体的产品，以及由任何两种以上的成分的组合、复合或聚集，或者由一种或多种成分的分解，或由一种或多种成分的其他类型的反应或相互作用直接地或间接地得到的任何产物。本发明中所述的“突变”包括氨基酸的置换、缺失、添加。
综上所述，本发明的突变体具有野生型FGF21没有的优点。这种优点包括改善的药理学效力和/或改善的药物稳定性。本发明的FGF21突变体蛋白具有一种或多种有利的生理学特征，包括在体内具有更显著的药效，具有更强热稳定性。另外，本发明的FGF21变体对2型糖尿病、肥胖、异常脂血症、或代谢综合症、或其任意的治疗具有潜在的作用。
附图简述
图1.SUMO-FGF21突变体表达载体示意图。
图2.纯化后SUMO-FGF21-LG核酸电泳图，M是DL5000DNA Marker，1是SUMO-FGF21-LG基因的纯化产物。
图3.纯化后SUMO-FGF21-AG核酸电泳图，M是DL5000DNA Marker，1是SUMO-FGF21-AG基因的纯化产物。
图4.纯化后pET22b(DE3)SUMO-FGF21-LG蛋白的SDS-PAGE图。
图5.纯化后FGF21-LG蛋白的SDS-PAGE图，M是蛋白分子量标准，1是FGF21-LG。
图6.纯化后FGF21-AG蛋白的SDS-PAGE图，M是蛋白分子量标准，1是FGF21-AG。
图7.FGF21-LG蛋白动物药效试验(降低血糖)结果图。
图8.FGF21-AG蛋白动物药效试验(降低血糖)结果图。
图9.FGF21-LG蛋白动物药效试验(对体重的影响)结果图。
图10.FGF21-AG蛋白动物药效试验(对体重的影响)结果图。
图11.FGF21-LG蛋白的热稳定性试验。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施中实验主要试剂耗材及仪器：克隆所用引物均为上海生工合成。聚合酶链式反应(PCR)中DNA聚合酶(primer star)、限制性内切酶、连接酶、Dpn I均购于Takara公司；DNA marker购于Thermo公司；普通DNA产物回收试剂盒、琼脂糖凝胶回收试剂盒购于TIANGEN公司。E.coliDH5α菌株，E.coliBL21菌株，pET22b-SUMO-FGF21质粒，SUMO蛋白酶购置北京索莱宝科技有限公司。Tris、咪唑购于上海生工，其他盐类购于国药，浓缩管购于Millipore。FPLC仪器使用的是GE公司的
Figure PCTCN2018082669-appb-000001
系统，使用的Ni-Sepharose层析柱和分子排阻层析 柱Hiload 16/60Superdex75pg均购于GE公司。BCA试剂盒购于Thermo。
表1.FGF21分子克隆所使用的引物
Figure PCTCN2018082669-appb-000002
a第1-8位为保护碱基，第9至14位为限制性内切酶位点；
b第1-3位为保护碱基，第4至9位为限制性内切酶位点。
实施例1：重组FGF21突变体表达载体的构建
以pET22b-SUMO-FGF21质粒为模板，第一次突变：利用上游引物171G-F和下游引物171G-R进行PCR定点突变。得到FGF21-P171G突变体。第二次突 变利用FGF21-P171G突变体为模板，以43C-F为上游引物和43C-R为下游引物进行PCR定点突变，得到FGF21-G43C-P171G突变体。第三次突变：利用FGF21-G43C-P171G突变体为模板，以31-32C-F为上游引物和31-32C-R为下游引物进行PCR定点突变，得到FGF21-AG突变体。第四次突变：以FGF21-AG为模板，以FGF21-LG-F为上游引物，以FGF21-LG-R为下游引物进行突变。得到FGF21-LG突变体。PCR反应条件如下。以上突变过程的PCR反应体系及反应程序如下：
PCR反应体系：2×Prime STAR HS(Premix)25μl，上游引物(10μM)1μl，下游引物(10μM)1μl，模板1μl，ddH 2O 22μl，总体积50μl。
PCR反应程序：第1步、预变性温度98℃10mins；第2步、变性温度98℃10secs；3、退火温度57℃5secs；第4步、延伸温度72℃，1min。从第2到4步重复30个循环。
每次突变后的产物用Dpn I酶在37℃酶切反应1h。将酶切产物进行琼脂糖凝胶电泳，并利用胶回收试剂盒回收质粒，取1ul质粒放入100ul的感受态细胞中，将细胞放入冰上30分钟，42℃热激90秒，加入400ul LB培养基，37℃振荡培养45分钟后涂平板，37℃倒置过夜培养。挑取单克隆作为模板，利用SUMO-F为上游引物和FGF21-R为下游引物进行PCR鉴定，其中FGF21-LG的鉴定结果如图2所示，FGF21-AG的鉴定结构如图3所示。经初步鉴定成功的FGF21突变体进行基因的测序与结果分析。样品由生工生物工程(上海)有限公司进行序列测定。采用DNAMAN软件进行序列分析。序列测定的结果：突变体FGF21-AG的核酸序列如SEQ ID NO.3所示，相应氨基酸序列如SEQ ID NO.4所示；突变体FGF21-LG的核酸序列如SEQ ID NO.5所示，相应氨基酸序列如SEQ ID NO.6所示。其构建载体示意图见图1。
将测序成功的含有编码FGF21突变体的核酸序列的质粒转化到BL21(DE3)菌株中，挑取单克隆菌株进行扩大培养，并将菌株保存在-80℃冰箱中。
实施例2：人重组FGF21突变体的表达
将FGF21突变体菌株按2％的接种量接种于5ml LB培养基(含100μg/ml氨苄青霉素)中，37℃、220rpm振荡培养过夜，取过夜培养菌液按2％接种于每瓶中，每2000ml三角瓶装500ml LB培养基(含100μg/ml氨苄青霉素)。培养到 OD 600为0.8时加入1.0M IPTG 500ul，在25℃诱导表达4个小时。4℃,8000r/min离心10分钟，弃上清，收集菌体，将菌体放于-80℃冻存。
实施例3：重组人FGF21突变体纯化
(1)重组人FGF21突变体初纯化
将表达得到的菌体用裂解缓冲液(50mM Tris,300mM NaCl,pH8.5)重悬，然后进行超声破碎(30％功率，超2秒，停5秒,总时间45分钟)，4℃14000rpm离心40分钟，收集上清液。将上清液加入已经用缓冲液预平衡的Ni-Sepharose层析柱，4℃旋转混合30分钟。将溶液穿出后，用5倍柱体积的缓冲液冲洗以去除未结合的蛋白和杂质，再用5倍柱体积的含30mM咪唑的缓冲液(50mM Tris,300mM NaCl,pH8.5)将非特异性的杂蛋白洗脱下来，再用5倍柱体积的含300mM咪唑的缓冲液(50mM Tris,300mM NaCl,pH8.5)将目的蛋白洗脱下来，用SDS-PAGE检测纯化结果(结果见图4)。
(2)重组人FGF21突变体酶切纯化
检测透析后的融合蛋白：以分子摩尔比为0.1％的比例加入SUMO酶，在4℃下酶切2h，向酶切后的蛋白溶液加入终浓度为20mM咪唑，然后加入到已经用缓冲液(50mM Tris,300mM NaCl,pH8.5)预平衡的Ni-Sepharose层析柱上，4℃旋转混合30分钟。收集流穿的溶液，用浓缩管将样品浓缩到10mg/ml，再经Superdex 75分子排阻层析柱纯化，分子排阻层析缓冲液(20mM PBS,100mM NaCl，pH7.2)，得到的蛋白即为FGF21突变体蛋白，实验中的FGF21及突变体如FGF21-LG和FGF21-AG蛋白都是用上述方法纯化。并经12％SDS-PAGE电泳检测，其中FGF21-LG蛋白的SDS-PAGE凝胶电泳结果如图5，FGF21-AG蛋白的SDS-PAGE凝胶电泳结果见图6。
实施例4：FGF21突变体降血糖实验
使用突变体蛋白进行动物降血糖实验。将ob/ob小鼠(购置于南京大学动物模式所)模型分成3组，每组8只，分别为载体对照组，即注射生理盐水，FGF21组和FGF21-LG组，实施方式为背部皮下注射，0.1mg/kg/天，连续给药七天，并且记录每天给药前的血糖值，如图7所示，FGF21-LG(如图7)组相对于野生型FGF21蛋白而言表现出更强的药效。同样，以FGF21-AG进行相同的实验， 实施方式为0.6mg/kg/天，并记录每天给药前的血糖值。实验说明FGF21-AG同样有很明显的降血糖作用，结果如图8所示。
实施例5：FGF21突变体降体重实验
使用FGF21突变体蛋白进行动物的降体重试验。将ob/ob小鼠分成4组，分别为载体对照组，即注射生理盐水，FGF21组和人FGF21-LG组，FGF21-AG组，每组8只。将纯化后的蛋白按照0.6mg/kg/天给药，连续给药6天，并且记录6天后体重变化，实验证实FGF21-LG(如图9)和FGF21-AG(如图10)组相对于野生型FGF21蛋白具有更明显的减轻体重作用，具有更强的药效。
实施例6：热稳定性试验
使用人FGF21突变蛋白进行实验，利用圆二色谱(CD)对FGF21及FGF21-LG进行变温实验，20-95℃，每5℃作为一个测试点进行测试。测试结果为FGF21的转化温度在69℃，而FGF21-LG的转化温度在98℃，实验证明人FGF21-LG相对于野生型FGF21具有更强的热稳定性(如图11)。同样，利用FGF21-AG，得到类似的CD结果。
可见，根据本发明的突变体与野生型相比具有更强的热稳定性。

Claims (10)

  1. 一种人FGF21突变体，其在野生型人FGF21蛋白氨基酸序列的基础上具有以下氨基酸改变：
    1)在野生型人FGF21蛋白氨基酸序列的第31位Ala和第32位His之间增加一个Cys，并且第43位Gly、第44位Ala中的任意1个或2个位置处的氨基酸突变为Cys，
    2)野生型人FGF21蛋白氨基酸序列的第171位Pro突变为Gly；和/或
    3)野生型人FGF21蛋白氨基酸序列的第24位至第31位的氨基酸片段用5-15个氨基酸，优选6-14个氨基酸，更优选7-13个氨基酸，还优选8-10个氨基酸，最优选8个氨基酸的片段替换。
  2. 根据权利要求1所述的突变体，其中用于所述替换的氨基酸片段为由式X 1X 2X 3X 4X 5X 6X 7X 8表示的8个氨基酸的片段，其中X 1-X 8各自独立地选自任意氨基酸，优选X 1是Ser或Asp，X 2是Gly或Asp，X 3是Pro或Ala，X 4是Ala或Gln，X 5是Gly或Gln，X 6是Leu或Tyr，X 7是Ser或His，和/或X 8是Ser或Ala。
  3. 根据权利要求2所述的突变体，其中用于所述替换的氨基酸片段的氨基酸序列是DDAQQTEA或SGPHGLSS。
  4. 根据权利要求3所述的突变体，其中所述突变体的氨基酸序列如SEQ ID NO：4或SEQ ID NO：6所示。
  5. 根据权利要求1所述的突变体，其中所述野生型人FGF21蛋白氨基酸序列如SEQ ID NO：1所示或是与SEQ ID NO：1具有90％以上，95％以上，优选98％以上，更优选99％以上的同源性的氨基酸序列。
  6. 核酸，其编码权利要求1-5中任一项所述的突变体。
  7. 载体，其包含权利要求6所述的核酸，所述载体优选为表达载体，更优选为原核表达载体。
  8. 宿主细胞，其包含权利要求7所述的载体。
  9. 药物组合物，其包含权利要求1-5中任一项所述的突变体以及药用载体。
  10. 权利要求1-5中任一项所述的突变体、权利要求6所述的核酸、权利要求7所述的载体或权利要求8所述的宿主细胞在制备药物中的用途，所述药物用于治疗2型糖尿病、肥胖、异常血脂症、或代谢综合征。
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