CN109836487B

CN109836487B - Human fibroblast growth factor 18, and soluble recombinant expression method, preparation and application thereof

Info

Publication number: CN109836487B
Application number: CN201910154313.5A
Authority: CN
Inventors: 曾鑫; 曹宇; 覃晓兰; 蔡春淞; 范玲玲; 范开
Original assignee: Peg Bio Biopharm Co ltd Chongqing
Current assignee: Peg Bio Biopharm Co ltd Chongqing
Priority date: 2019-03-01
Filing date: 2019-03-01
Publication date: 2022-06-17
Anticipated expiration: 2039-03-01
Also published as: CN109836487A

Abstract

The invention discloses a human fibroblast growth factor 18, the amino acid sequence of which is shown as SEQ ID NO 1; the soluble recombinant expression method of the human fibroblast growth factor 18 with the deletion of the N terminal comprises the following steps: (1) selecting a plasmid containing a T7 promoter as an expression vector and a corresponding escherichia coli as a host bacterium; (2) 1 and a recombinant and an expression strain of cDNA corresponding to SEQ ID NO; (3) the induction expression conditions of the expression strain are as follows: the temperature is 15-30 ℃, and the induction and culture time is 4-16 hours under the induction of lactose or IPTG. The preparation method of the human fibroblast growth factor 18 with the deleted N terminal, the preparation of the human fibroblast growth factor 18 with the deleted N terminal and the application of the human fibroblast growth factor 18 with the deleted N terminal in treating osteoarthritis or articular cartilage damage.

Description

Human fibroblast growth factor 18, and soluble recombinant expression method, preparation and application thereof

Technical Field

The invention relates to the field of biological pharmacy, in particular to human fibroblast growth factor 18 and a soluble recombinant expression method, a preparation and application thereof.

Background

Fibroblast Growth Factors (FGFs) are structurally related proteins encoded by FGF gene family members, have the effects of promoting DNA synthesis and cell division for cells derived from various mesoderms and neuroectoderms, such as endothelial cells, fibroblasts, smooth muscle cells and the like, are mainly secreted by the fibroblasts, the endothelial cells, osteocytes and inert cells, and have a relative molecular weight of generally 17-34 kD. Fibroblast growth factor 18 (hereinafter, abbreviated as FGF18) was first isolated from mouse embryos by Ohbayashi, a japanese scientist, 1998, and plays an important role in the development of bone growth.

It is reported in the literature (Ornitz and Marie, Genes & Development,16: 1446-.

FGF18 (trade name Sprifermin), developed by Merck Serono, elicits stimulation of bone matrix synthesis and chondrocyte turnover by chondrocytes. An international multicenter phase 2 clinical study (FORWARD study) reported by the ACR congress in 2017 discusses the curative effect of Sprifermin injection in the bone joint on OA of the knee joint, and the result shows that Sprifermin injection in the joint can effectively prevent the loss of the joint cartilage. After 2 years of treatment, the thickness of the tibial articular cartilage of the Spriformin group is increased in dosage tolerance, the thickness of the cartilage of the 100mg group every 6 months and the cartilage of the 100mg group every 12 months is respectively increased by 0.03mm and 0.02mm, while the thickness of the cartilage of the placebo group is reduced by 0.02mm (P <0.001), the WOMAC score of each treatment group is improved by 50%, the safety of the medicine is good during the research period, and the incidence rate of adverse reactions of patients in each group is similar. Phase ii clinical studies of spifermin for the treatment of gonarthritis have been successfully completed and are in progress in phase iii clinical studies.

The patent (US7858341) discloses that the defect of complicated process is that FGF18 is obtained by using genetic engineering technology, a prokaryotic expression system of escherichia coli and an inclusion body form to express inactive FGF18, and then the solution, renaturation and column chromatography are carried out by using a denaturant.

FGF18 is a heparin-binding protein, its isoelectric point is 9.86, its amino acid sequence contains a lot of basic amino acids (lysine and arginine), its proportion is up to 20.6%, and a lot of negatively charged amino acids are aggregated and mutually repelled, so that the peptide bond is unstable, and its N-terminal is easily broken, so that it is very unstable for use in pharmaceutical preparation. The structures of FGF18 reported in the literature or the patent at present all belong to basic proteins, the structures are unstable, and the N end is easy to break; the preparation method is characterized in that the expression is carried out in the form of inclusion bodies in the escherichia coli, disulfide bonds cannot be formed in cells, the pretreatment is complex, the renaturation conditions are harsh, and the industrial production difficulty is high.

Disclosure of Invention

In order to achieve the above object, the present invention provides a human fibroblast growth factor 18, wherein the human fibroblast growth factor 18 has a stable N-terminus, thereby reducing the problem that the N-terminus of the growth factor 18 in the prior art is easily broken; in addition, the invention provides a soluble expression method and a preparation method of the human fibroblast growth factor 18, which simplify the preparation means and improve the preparation efficiency, the method is simple and easy to popularize, and the obtained N-terminal deletion human fibroblast growth factor 18 can be used for treating osteoarthritis or articular cartilage injury.

In order to achieve the purpose, the invention adopts the following technical scheme:

a human fibroblast growth factor 18 has an amino acid sequence shown in SEQ ID NO. 1.

A soluble recombinant expression method of human fibroblast growth factor 18 comprises the following steps:

(1) selecting a plasmid containing a T7 promoter as an expression vector and a corresponding escherichia coli as a host bacterium;

(2) 1 and a recombinant and an expression strain of cDNA corresponding to SEQ ID NO;

(3) the induction expression conditions of the expression strain are as follows: the temperature is 15-30 ℃, and the induction and culture time is 4-16 hours under the induction of lactose or IPTG.

A preparation method of human fibroblast growth factor 18 comprises the following steps:

(1) homogenizing under pressure, crushing at low temperature, centrifuging, and collecting supernatant;

(2) capturing FGF18 delta N in cell disruption supernatant by anion chromatography;

(3) copper ions are used as a disulfide bond correct pairing accelerator;

(4) after correct pairing of disulfide bonds, the target product is captured by heparin affinity chromatography.

A preparation of human fibroblast growth factor 18, comprising by weight: 5-50 ug/dose of FGF18 delta N, 0.1-0.5 mg/dose of surfactant poloxamer, 10-50 mg/dose of stabilizer sucrose, 1.0-5.0 mg/dose of N-acetyl glucuronic acid repeatedly and alternately formed sodium hyaluronate and 400-800 ug/dose of antioxidant histidine.

Furthermore, the pH range of the preparation of the human fibroblast growth factor 18 with the deletion of the N end is 6.50-8.50.

The human fibroblast growth factor 18 with the N-terminal deletion is used for treating osteoarthritis or articular cartilage damage.

Compared with the prior art, the invention has the following beneficial effects:

the N-terminal of the human fibroblast growth factor-deleted N-terminal disclosed by the invention is deleted for 18, namely the N-terminal is deleted for 12 amino acids, so that the structure is stable, and the biological activity is kept; the soluble expression of the N-terminal deletion human fibroblast growth factor disclosed by the invention has the advantages that the disulfide bond pairing rate can reach more than 90% in a short time without a deforming agent, the process is simple, and the industrial production is facilitated; the invention discloses a preparation with an N-terminal deleted human fibroblast growth factor 18, which maintains the activity of the N-terminal deleted human fibroblast growth factor 18 and is used for treating osteoarthritis or articular cartilage injury.

Drawings

FIG. 1: a schematic diagram of the construction of pET-30a-FGF18 delta N recombinant plasmid;

FIG. 2: pET-30a-FGF18 delta N restriction enzyme identification picture, wherein Lane 1 is pET-30a-FGF18 delta N recombinant plasmid with correct restriction enzyme identification;

FIG. 3: SDS-PAGE of FGF18, pre-column chromatographed FGF 18. DELTA.N protein in the left lane; the middle lane is FGF18 Δ N protein after column chromatography;

FIG. 4: an RP-HPLC detection map before FGF18 renaturation shows that the peak 16.098min is FGF18 delta N before renaturation;

FIG. 5: an RP-HPLC detection map shows that after FGF18 renaturation, the 14.097min peak is FGF18 delta N after renaturation;

FIG. 6: 72H fragmentation electrophoretic detection images of FGF18 and FGF18 delta N in a 37 ℃ drug inspection box;

FIG. 7 is a schematic view of: EC50 ratios of FGF18 and FGF18 Δ N to stimulate proliferation of BaF3 cells that have been transfected with the FGFR3 iiic receptor gene;

FIG. 8: graph comparing right bone joint thickening after treatment of model meniscus amputated Lewis rats with FGF18 and FGF18 Δ N: vehicle control Vehicle group (G1), FGF18 treatment group (G2), FGF18 Δ N treatment group (G3);

FIG. 9: morphological observations compare graphs after treatment of model meniscus-dissected Lewis rats with FGF18 and FGF18 Δ N: vehicle control Vehicle group (G1), FGF18 treatment group (G2), FGF18 Δ N treatment group (G3);

FIG. 10: model of meniscal dissection Lewis rats treated with FGF18 and FGF18 Δ N, hematoxylin-eosin staining profile of pathological sections: vehicle control Vehicle group (G1), FGF18 treatment group (G2), FGF18 Δ N treatment group (G3);

FIG. 11: after treatment of model meniscus-dissected Lewis rats with FGF18 and FGF18 Δ N, pathological sections were stained with safranin fast green: vehicle control Vehicle group (G1), FGF18 treatment group (G2), FGF18 Δ N treatment group (G3).

Detailed Description

The present invention will be described in further detail below by way of specific embodiments:

a human fibroblast growth factor 18 is obtained by deleting partial amino acids from the N-terminal of the human fibroblast growth factor 18 (hereinafter referred to as FGF18) so as to obtain the human fibroblast growth factor 18 with a stable N-terminal, which is hereinafter referred to as FGF18 delta N.

(3) copper ions are used as a disulfide bond correct pairing promoter;

The preparation method of the human fibroblast growth factor 18 disclosed by the invention comprises the following specific steps of:

s1 selects an expression system containing a T7 promoter as an expression plasmid vector and corresponding escherichia coli as host bacteria;

s2 selecting two suitable endonucleases, respectively performing double enzyme digestion on a plasmid vector and N-terminal deletion of the gene of the human fibroblast growth factor 18, connecting to construct a recombinant plasmid, and transferring the recombinant plasmid into an escherichia coli host bacterium to screen a recombinant expression strain;

s3 transferring the recombinant expression strain to a culture medium for fermentation culture at a certain temperature, and inducing and expressing the target protein by using a proper inducer;

s4 anion chromatography captures the precursor, a disulfide bond pairing promoter is supplemented, disulfide bond pairing is correct in a short time, and the FGF18 delta N protein with biological activity is obtained by heparin affinity chromatography;

the plasmid vectors described in step S1 are pET-30a and pET-30c, preferably pET-30 a.

The expression host bacterium described in step S1 is escherichia coli, more specifically, e.coli BL21, e.coli BL21(DE3), e.coli BL21(DE3) plysS, preferably e.coli BL21(DE3) pllyss.

The endonuclease described in step S2 is NdeI endonuclease at the 5 'end and BamHI endonuclease at the 3' end, respectively.

The culture medium in the step S3 is selected from a culture medium suitable for growth of Escherichia coli, and can be LB, M9CA, M9-YE-Glu culture medium or a modification and combination thereof on the basis of the culture medium.

The culture temperature in step S3 is 20-37 deg.C, preferably 37 deg.C.

The inducer of step S3 can be lactose, galactose or IPTG (isopropyl-. beta. -D-thiogalactoside), preferably IPTG.

The concentration of the inducer IPTG in the step S3 can be 0.1 mM-2 mM, and preferably 0.2 mM.

The culture time in step S3 may be 4-16 h, preferably 6 h.

The renaturation accelerator described in step S4 is cystine/cysteine, reduced Glutathione (GSH)/oxidized glutathione (GSSG), DTT/GSSG, copper ion, preferably copper ion.

In addition, the patent (WO2012172072) discloses a lyophilized formulation comprising FGF18, including FGF18, a buffer, a poloxamer surfactant and sucrose as stabilizers, the lyophilized formulation of FGF18 showing promising results in the treatment of osteoarthritis or cartilage damage.

Osteoarthritis (OA): degenerative arthritis, proliferative osteoarthritis, is a chronic joint disease characterized by degeneration, destruction, and hyperosteogeny of articular cartilage, and the incidence of osteoarthritis increases with age, a common joint disease in the elderly. The treatment of osteoarthritis includes drug therapy, surgical therapy, and non-drug therapy. The drug therapy comprises glucosamine (GlcNH2), non-steroidal analgesic and anti-inflammatory drugs (NSAIDs), Chondroitin Sulfate (CS), and sodium Hyaluronate (HA).

Patent (CN106029085) discloses hydrogel of xyloglucan containing FGF18 and preparation method thereof, which is used for treating cartilage diseases such as osteoarthritis or cartilage injury. In the treatment of diseases of cartilage damage, directional injection of a hydrogel comprising FGF18 and xyloglucan to the site of cartilage damage promotes cartilage growth in the damaged area without inducing uncontrolled growth of normal cartilage in otherwise healthy areas.

In preparing a pharmaceutical composition comprising a biologically active protein, the preparation must be such that the activity of the protein is maintained for a suitable period of time. Protein fragmentation, denaturation, aggregation, and oxidation may cause loss of activity and stability. While excipients and protective agents can improve the stability of proteins, the stabilizing effect of these excipients is highly dependent on the polymer in the pharmaceutical formulation, the nature of the excipient, and the biologically active protein itself.

Sodium Hyaluronate (Sodium Hyaluronate), also known as Sodium Hyaluronate, is a macromolecular polysaccharide biomaterial formed by the repeated alternation of N-acetylglucosaminic acid. Sodium hyaluronate is a main component of joint synovial fluid, is one of components of cartilage matrixes, has various physiological functions of participating in electrolyte and water regulation in extracellular fluid, lubricating joints, resisting infection, participating in wound healing and the like, plays an important role in protecting, nourishing and playing functions of joints, and mainly shows lubrication, covering barrier and buffering stress of joint cavities, and has good curative effect on mild and moderate knee osteoarthritis. Because sodium hyaluronate is basically a chemical inert substance and has compatibility with most chemical substances, sodium hyaluronate can be used as a medium for a plurality of medicines in eye drops, and has the effects of synergism, slow release and reduction of side effects of the medicines. In the future, sodium hyaluronate is used as a medium for orthopedic and ophthalmic medicines, is added into antitumor medicines, non-steroidal anti-inflammatory analgesic medicines and glucocorticoids, and also has obvious medicine synergism and slow release effects.

Therefore, the human fibroblast growth factor 18 disclosed by the invention can be used as a raw material to research and develop a dosage form with better performance and higher biological safety for treating osteoarthritis or cartilage injury.

The invention discloses a preparation of human fibroblast growth factor 18, which comprises the following components in percentage by weight: 5-50 ug/dose of FGF18 delta N, 0.1-0.5 mg/dose of surfactant poloxamer, 10-50 mg/dose of stabilizer sucrose, 1.0-5.0 mg/dose of N-acetyl glucuronic acid repeatedly and alternately formed sodium hyaluronate and 400-800 ug/dose of antioxidant histidine.

Wherein, the content of FGF18 delta N is preferably 30 ug/dose; poloxamer, preferably in an amount of 0.2 mg/dose; the content of the stabilizer sucrose is preferably 30 mg/dose; the content of sodium hyaluronate formed by N-acetylglucosaminic acid repeatedly and alternately is preferably 2.5 mg/dose; the antioxidant histidine content is preferably 775 ug/dose.

The pH range of the preparation of the human fibroblast growth factor 18 is 6.50-8.50; the pH range of the preparation is preferably 7.0-7.5; most preferably 7.20.

The human fibroblast growth factor 18 is used for treating osteoarthritis or articular cartilage damage.

Example 1 construction and inducible expression of genetically engineered Strain with N-terminal deletion of human fibroblast growth factor 18

A cDNA sequence (SEQ ID NO:2) designed based on the N-terminal deletion of the amino acid sequence (SEQ ID NO:1) of human fibroblast growth factor 18. The whole gene was synthesized and inserted into a vector plasmid pUC-57 to obtain a plasmid pUC-57-FGF 18. delta.N. The pET-30a-FGF delta N recombinant plasmid is constructed by connecting FGF18 delta N gene with a vector plasmid pET-30a by using NdeI and BamHI restriction enzyme cutting sites, the plasmid is transformed into Escherichia coli expression host bacteria BL21(DE3) PlysS, and the PlysS recombinant engineering bacteria pET-30a-FGF18 delta N/BL21(DE3) are constructed through expression screening. Streaking and inoculating the engineering strain, selecting the lawn, inoculating the lawn in a test tube containing a culture medium, activating, transferring the lawn into a triangular agent, and culturing overnight to obtain the seed liquid in the tank. Inoculating the seed solution in the upper tank into a 30L fermentation tank, culturing at 37 deg.C, regulating rotation speed, air flow amount, and pure oxygen flow amount to maintain dissolved oxygen > 30%, and maintaining pH at 7.0. When the OD600 of the bacterial liquid is 10.0, 0.2mM IPTG is added to the bacterial liquid, the fermentation is stopped after the culture is continued for 12 hours, and the bacterial cells are collected by centrifugation.

Example 2 disulfide bond formation with N-terminal deletion of human fibroblast growth factor 18

After the thalli is resuspended, homogenizing under high pressure and breaking the thalli, capturing FGF18 delta N by anion chromatography, diluting with a diluent (20mM Tris, pH7.5) in an equal volume ratio of 2:1, adding copper ions with a final concentration of 5uM, stirring at a low speed of 4 ℃ for 4h, and detecting by HPLC (high performance liquid chromatography), wherein the disulfide bond pairing rate is not lower than 90%, and the FGF18 delta N with high activity is obtained.

Example 3N-terminal deletion of human fibroblast growth factor 18 preparation

Weighing a proper amount of poloxamer, sucrose and histidine, dissolving to prepare an auxiliary material solution I with a certain concentration; weighing a proper amount of hyaluronic acid, swelling, and sterilizing with high pressure steam to obtain a solution II;

diluting FGF18 delta N protein solution to a certain concentration by using water for injection to obtain solution III;

mixing the solution I and the solution III according to a certain volume ratio, and performing sterile filtration to obtain a solution IV;

mixing the solution II and the solution IV in equal volume, and subpackaging in small dose of 0.5 ml/dose, wherein the pH value is 7.20;

and (3) freeze drying: pre-freezing at-45 deg.C for 180 min; sublimation drying in the first stage at-45-20 deg.C for 10h and-20-10 deg.C for 10h while maintaining vacuum degree of 0.8 mbar; and (4) carrying out resolution drying at the temperature of-10 ℃ for 10 h.

Each dose contains FGF18 delta N30 ug, poloxamer 188200ug, hyaluronic acid 2.5mg, sucrose 30mg, and histidine 775.8 ug.

Example 4 stability Studies of N-terminal deletion of human fibroblast growth factor 18

FGF18 and FGF18 delta N freeze-dried powder are dissolved in water for injection, are added into sterile 3ml of penicillin (1 ml/piece), are placed in a light-shielding 37 ℃ medicine inspection box, are respectively sampled at 12H, 24H, 36H, 48H, 60H and 72H, and are subjected to SDS-PAGE detection, FGF18 gradually starts to degrade at 12H, is completely degraded at 60H, and FGF18 delta N does not degrade at 72H.

Example 5 in vitro Activity assay for N-terminal deletion of human fibroblast growth factor 18

BaF3 cells transfected with FGFR3 IIIc receptor gene, centrifuged at 1000rmp for 5min, washed 3 times with RPMI 1640, resuspended in RPMI 1640 medium containing 10% FCS (without IL-3), counted, and added at 2-2.5X 105 cells/ml/well with 100. mu.l, 37 ℃ C., 5% CO₂The culture was carried out for 26 hours. The recombinant human FGF18 standard substance and FGF18 delta N are diluted by a living culture solution (containing a culture medium 1640, 10% newborn bovine serum albumin and 1ug/ml heparin sodium) by 5 times (the concentration range is 0.002U/ml-177U/ml) for 8 dilutions, 100 mu L of each well is added, multiple wells are set, the culture is carried out at 37 ℃ and 5% CO2 for 48 hours, 10 mu L of ATPLite 1step is added after 48 hours, and the result is measured by a fluorescence microplate reader. According to the EC50 ratio of the recombinant human FGF18 standard and FGF18 delta N, the results show that the activities of the two are not different.

Example 6 application of N-terminal deletion of human fibroblast growth factor 18 in rat knee osteoarthritis model caused by meniscal transection

Male Lewis rats with the body weight of 320-380G are randomly divided into three groups, namely a Vehicle control Vehicle group (G1), an FGF18 treatment group (G2) and an FGF18 delta N treatment group (G3), and each group is 8. The right knee joint of each group of rats was surgically cut through the entire thickness of the medial meniscus to simulate a complete tear before treatment began, inducing the formation of gonarthritis, and the left knee joint was controlled in a sham surgical manner (consistent surgical treatment, so as not to cut the medial meniscus). Administration via intra-articular route is started 21 days after operation, and 50 mul/joint solvent is administered to rats in the solvent control group every week for 3 times; FGF 18-treated group rats were administered 3 μ g/joint FGF18 weekly for 3 consecutive doses; the FGF18 delta N treatment group rats are administered 3 mu g/joint FGF18 delta N for 3 times per week, after treatment (namely 3 weeks after treatment), the right knee joint is collected by conventional dissection for histopathological evaluation of potential pharmacological activity, and the histological observation of knee joint cartilage lesions is subjected to semi-quantitative scoring by using an OARSI scoring system. The results show that the repair tissues generated in the vehicle control group are mainly mixed fibrocartilage (i.e. collagen type i and type ii, with a small amount of tissue type iv collagen staining), and the repair tissues of the FGF18 treated group and the FGF18 Δ N treated group are positive for type ii collagen and have clear extracellular peripheral type iv collagen staining, showing mature hyaline cartilage repair, demonstrating no difference in efficacy between the FGF18 treated group and the FGF18 Δ N treated group.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

SEQUENCE LISTING

<110> Chongqing Paijin Biotechnology Co., Ltd

<120> a novel human fibroblast growth factor 18 and soluble recombinant expression method, preparation method and preparation method thereof

Agent and use

<130> CQ201910010

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 159

<212> PRT

<213> Artificial sequence

<400> 1

Met Asn Gln Thr Arg Ala Arg Asp Asp Val Ser Arg Lys Gln Leu Arg

1 5 10 15

Leu Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His Ile Gln Val Leu

20 25 30

Gly Arg Arg Ile Ser Ala Arg Gly Glu Asp Gly Asp Lys Tyr Ala Gln

35 40 45

Leu Leu Val Glu Thr Asp Thr Phe Gly Ser Gln Val Arg Ile Lys Gly

50 55 60

Lys Glu Thr Glu Phe Tyr Leu Cys Met Asn Arg Lys Gly Lys Leu Val

65 70 75 80

Gly Lys Pro Asp Gly Thr Ser Lys Glu Cys Val Phe Ile Glu Lys Val

85 90 95

Leu Glu Asn Asn Tyr Thr Ala Leu Met Ser Ala Lys Tyr Ser Gly Trp

100 105 110

Tyr Val Gly Phe Thr Lys Lys Gly Arg Pro Arg Lys Gly Pro Lys Thr

115 120 125

Arg Glu Asn Gln Gln Asp Val His Phe Met Lys Arg Tyr Pro Lys Gly

130 135 140

Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr Thr Val Thr Lys

145 150 155

<210> 2

<211> 492

<212> DNA

<213> Artificial sequence

<400> 2

catatgaacc agacccgtgc gcgtgacgat gtgagccgta agcagctgcg tctgtaccaa 60

ctgtatagcc gtaccagcgg caaacacatc caagttctgg gtcgtcgtat tagcgcgcgt 120

ggtgaagacg gcgataaata cgcgcagctg ctggttgaga ccgacacctt cggcagccaa 180

gttcgtatca agggtaaaga gaccgaattt tatctgtgca tgaaccgtaa gggtaaactg 240

gtgggcaagc cggatggtac cagcaaagaa tgcgtgttca ttgagaaggt tctggaaaac 300

aactacaccg cgctgatgag cgcgaaatac agcggctggt acgttggttt taccaagaaa 360

ggccgtccgc gtaagggtcc gaaaacccgt gagaaccagc aagatgttca cttcatgaag 420

cgttacccga aaggccagcc ggaactgcaa aagccgttta aatataccac cgttaccaag 480

taatgaggat cc 492

Claims

1. A human fibroblast growth factor 18, comprising: the amino acid sequence is shown in SEQ ID NO. 1.

2. A method of soluble recombinant expression of human fibroblast growth factor 18 according to claim 1, wherein: the method comprises the following steps:

(1) selecting a plasmid containing a T7 promoter as an expression vector and taking escherichia coli as host bacteria;

(2) constructing a recombinant plasmid containing a nucleic acid molecule of an amino acid sequence corresponding to the code SEQ ID NO. 1, transferring the recombinant plasmid into escherichia coli host bacteria, and screening a recombinant expression strain;

(3) inducing the expression of the recombinant expression strain, wherein the conditions for inducing the expression are as follows: the temperature is 15-30 ℃, and the induction and culture time is 4-16 hours under the induction of lactose or IPTG.

3. A method of preparing human fibroblast growth factor 18 according to claim 1, wherein: the method comprises the following steps:

(1) homogenizing under pressure, crushing thallus at low temperature, centrifuging, and collecting supernatant;

(2) capturing the human fibroblast growth factor 18 in the supernatant by anion chromatography;

(3) a disulfide bond pairing accelerator is supplemented;

(4) after the disulfide bonds in the human fibroblast growth factor 18 are correctly paired, heparin affinity chromatography is carried out to capture the target product human fibroblast growth factor 18;

wherein the thallus is a bacterial strain expressing the human fibroblast growth factor 18, and the disulfide bond pairing accelerator is copper ions.

4. A formulation comprising human fibroblast growth factor 18 according to claim 1, wherein: comprises the following components by weight: 5-50 ug/dose of the human fibroblast growth factor 18, 0.1-0.5 mg/dose of surfactant poloxamer, 10-50 mg/dose of stabilizer sucrose, 1.0-5.0 mg/dose of N-acetylglucosaminic acid sodium hyaluronate formed by repeated alternation, and 400-800 ug/dose of antioxidant histidine.

5. The preparation of human fibroblast growth factor 18 according to claim 4, wherein: the pH range of the preparation is 6.50-8.50.

6. Use of human fibroblast growth factor 18 according to claim 1 in the preparation of a formulation for the treatment of osteoarthritis or articular cartilage damage.