CN111303298B

CN111303298B - Fusion protein containing phosphatase, and product and application thereof

Info

Publication number: CN111303298B
Application number: CN202010103271.5A
Authority: CN
Inventors: 李瑛�
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-02-19
Filing date: 2020-02-19
Publication date: 2023-03-21
Anticipated expiration: 2040-02-19
Also published as: CN111303298A

Abstract

The invention relates to the field of biomedicine, and particularly provides a fusion protein containing phosphatase, and a product and application thereof. The fusion protein containing phosphatase provided by the invention comprises phospholipase, connecting peptide and amphipathic protein, wherein the phospholipase and the amphipathic protein are connected through the connecting peptide. The invention adopts a fusion protein technology to couple phospholipase and amphipathic protein from food-borne fungi, overcomes the defect that the phospholipase cannot be absorbed orally, has stable structure and oral absorption effect, can greatly increase liver accumulation of the phospholipase so as to achieve the effect-taking dose Cmax of the phospholipase, and increases the stability of a medicament in vivo. In addition, the fusion protein shows good NASH treatment function in the bodies of experimental animals, and is beneficial to reducing drug intake, thereby reducing medical cost.

Description

Fusion protein containing phosphatase, and product and application thereof

Technical Field

The invention relates to the field of biomedicine, in particular to a fusion protein containing phosphatase, and a product and application thereof.

Background

Nonalcoholic steatohepatitis (NASH), an extremely advanced form of nonalcoholic fatty liver disease (NAFLD), is defined as the appearance of steatosis with inflammation and hepatocellular injury. NASH can lead to late stage liver fibrosis, cirrhosis, liver failure, and the development of liver tumors. NASH has doubled its onset over the last 20 years and is now the most common liver disease in western countries. The major risk factors for NASH include obesity, type 2 diabetes (T2 DM), and dyslipidemia and metabolic syndrome. In the united states, NAFLD accounts for approximately 10-46% of the general population, with approximately 10-30% of patients developing NASH. Currently, NASH-related cirrhosis is the third most common cause of liver transplantation in the united states, and is expected to be the leading cause in 2020. However, despite the urgent medical need, to date the FDA has not approved any drug for NASH treatment. Currently, diagnosis of NASH relies on liver biopsy assessment and treatment is aimed at controlling the progression of diseases associated with obesity, type 2 diabetes, and hyperlipidemia.

Phospholipase (rOLY) has the function of interfering the differentiation of adipose stem cells, can stimulate the differentiation of white adipocytes into brown adipocytes, and studies have confirmed that phospholipase (rOLY) can interfere the mitochondrial function of adipocytes at the cellular level. The results of the above studies fully confirm the effect of phospholipase (rooly) on NASH diseases, but the efficacy of phospholipase (rooy) in experimental animals has not been confirmed all the time, and there is no efficacy or unstable efficacy in experimental animals after administration of phospholipase (rooy), so there is no effective means for how phospholipase (rooly) is used to treat NASH diseases.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

In the research process, the inventor analyzes the reasons of no drug effect and unstable drug effect of the phospholipase in the test animal body by adopting a tissue biochemical method, and finds that the distribution of the liver in the tissue distribution result of the phospholipase after injection administration is only 0.2-0.3 percent and is greatly lower than the effective dose of the phospholipase (in vitro experiment result). This means that researchers need to raise the injected dose by thousands of times to obtain good therapeutic results from experimental animal models, however, such large doses of injected drugs are clearly undesirable for treating NASH disease.

Therefore, the inventors thought that increasing liver accumulation of phospholipase drugs is a means to increase the therapeutic coefficient of phospholipase. Therefore, the technical scheme of the invention is provided, and the purpose is achieved by utilizing the liver first-pass effect of the oral medicament.

First pass effect of liver: the medicine needs to be orally taken, and is absorbed by small intestine after being orally taken. Drugs absorbed by the small intestine do not immediately enter the peripheral blood, but enter the liver in large quantities for metabolism.

In order to achieve the above purpose of the present invention, the following technical solutions are adopted:

a phosphatase-containing fusion protein comprising a phospholipase, a linker peptide and an amphipathic protein, the phospholipase and amphipathic protein being linked by the linker peptide.

Further, the amino acid sequence of the phospholipase is as follows:

AYAQWVIILIHNVGQQNVKIKNLNASWGKLYADGDKDTEVPASKYEGMVIAPGDQVQINACGREDAAEGTTGTFDLVDPNDSDKQVRHISWDCPWGTKANSWVVGGSNSKWMIEYTGQNLDSGALGTITVNTLKIGN(SEQID NO.1)；

preferably, the amino acid sequence of the connecting peptide is shown in SEQ ID NO.2 or SEQ ID NO. 3:

SGTPTPTPTPTGEF(SEQ ID NO.2)；

GGGSGGGSGGGS(SEQ ID NO.3)；

preferably, the amino acid sequence of the amphiphilic protein is shown as SEQ ID NO.4:

MKFFTAAALFAAVAIAGPVEVRTGGGSICPDGLFSNPQCCDTQLLGIIGLGCEVPSQTPRDGADFKNICAKTGDQALCCVLPIAGQDLLCQAAVGAA(SEQ ID NO.4)；

further, the fusion protein comprises the following:

phospholipase shown in SEQ ID NO.1, connecting peptide shown in SEQ ID NO.2 and amphipathic protein shown in SEQ ID NO. 4;

or the amphipathic protein shown in SEQ ID NO.4, the connecting peptide shown in SEQ ID NO.3 and the phospholipase shown in SEQ ID NO. 1;

further, the amino acid sequence of the fusion protein is:

AYAQWVIILIHNVGQQNVKIKNLNASWGKLYADGDKDTEVPASKYEGMVIAPGDQVQINACGREDAAEGTTGTFDLVDPNDSDKQVRHISWDCPWGTKANSWVVGGSNSKWMIEYTGQNLDSGALGTITVNTLKIGNSGTPTPTPTPTGEFMKFFTAAALFAAVAIAGPVEVRTGGGSICPDGLFSNPQCCDTQLLGIIGLGCEVPSQTPRDGADFKNICAKTGDQALCCVLPIAGQDLLCQAAVGAA(SEQ ID NO.5)；

or the like, or a combination thereof,

MKFFTAAALFAAVAIAGPVEVRTGGGSICPDGLFSNPQCCDTQLLGIIGLGCEVPSQTPRDGADFKNICAKTGDQALCCVLPIAGQDLLCQAAVGAAGGGSGGGSGGGSAYAQWVIILIHNVGQQNVKIKNLNASWGKLYADGDKDTEVPASKYEGMVIAPGDQVQINACGREDAAEGTTGTFDLVDPNDSDKQVRHISWDCPWGTKANSWVVGGSNSKWMIEYTGQNLDSGALGTITVNTLKIGN(SEQ ID NO.6)。

the fusion protein-related biomaterial is any one of the following biomaterials:

(a) A nucleic acid molecule encoding a fusion protein;

(b) An expression cassette comprising the nucleic acid molecule of (a);

(c) A recombinant vector comprising the nucleic acid molecule of (a) or the expression cassette of (b);

(d) A recombinant expression system comprising the nucleic acid molecule of (a), the expression cassette of (b), or the recombinant vector of (c).

The preparation method of the fusion protein comprises the steps of introducing the nucleic acid molecule for coding the fusion protein into a host to obtain a recombinant expression system, and expressing the recombinant expression system to obtain the fusion protein;

preferably, the nucleic acid molecule encoding the fusion protein is enzymatically linked with an expression plasmid pPIC9 to obtain a recombinant expression vector, yeast is transfected, and the fusion protein is obtained by expression.

The fusion protein is applied to the preparation of the NASH treatment medicine.

A medicament for treating NASH comprises the fusion protein and optional pharmaceutically acceptable auxiliary materials.

Further, the effective amount of the fusion protein is 10-30 mg/person/day;

preferably, the auxiliary material comprises at least one of water-soluble filler auxiliary material, pH regulator, stabilizer, water for injection or osmotic pressure regulator;

preferably, the water-soluble filler adjuvant comprises at least one of mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose or galactose;

preferably, the pH adjusting agent comprises citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, potassium bicarbonate, or ammonium bicarbonate;

preferably, the stabilizer includes at least one of EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, sodium lauryl sulfate, or tris (hydroxymethyl) aminomethane;

preferably, the stabilizer comprises at least one of sodium metabisulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000 and tris (hydroxymethyl) aminomethane;

preferably, the tonicity modifier comprises sodium chloride and/or potassium chloride;

preferably, the administration mode of the NASH treatment drug is oral administration;

preferably, the dosage form of the NASH treatment drug is freeze-dried powder.

A method for preparing a medicament freeze-dried powder for treating NASH comprises the steps of uniformly mixing fusion protein and optional pharmaceutically acceptable auxiliary materials, and freeze-drying to obtain the medicament freeze-dried powder, tablets, capsules, oral liquid or granules for treating NASH.

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

the invention provides a fusion protein containing phosphatase, which comprises phospholipase, connecting peptide and amphipathic protein, wherein the phospholipase and the amphipathic protein are connected through the connecting peptide. The invention adopts the fusion protein technology to couple the phospholipase and the amphipathic protein from the food-borne fungi, overcomes the defect that the phospholipase cannot be absorbed orally, has the functions of stabilizing the structure and absorbing orally, can greatly increase the liver accumulation of the phospholipase so as to achieve the effect-taking dose Cmax of the phospholipase, and increases the stability of the medicine in vivo. In addition, the fusion protein shows good NASH treatment function in the bodies of experimental animals, and is beneficial to reducing drug intake, thereby reducing medical cost.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a silver stained gel of the fusion protein (SEQ ID NO. 5) in example 1 of the present invention, wherein the Lane 1;

FIG. 2 is an HPLC chromatogram of the fusion protein (SEQ ID NO. 6) in example 1 of the present invention;

FIG. 3 shows the results of MFN2 activation and its phosphorylation by the fusion protein (SEQ ID NO. 5) in example 6 of the present invention.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.

Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.

The invention provides a fusion protein containing phosphatase, which comprises phospholipase, connecting peptide and amphipathic protein, wherein the phospholipase and the amphipathic protein are connected through the connecting peptide.

The invention adopts the fusion protein technology to couple the phospholipase and the amphipathic protein from the food-borne fungi, overcomes the defect that the phospholipase cannot be absorbed orally, has the functions of stabilizing the structure and absorbing orally, can greatly increase the liver accumulation of the phospholipase so as to achieve the effect-taking dose Cmax of the phospholipase, and increases the stability of the medicine in vivo. In addition, the fusion protein shows good NASH treatment function in the bodies of experimental animals, and is beneficial to reducing drug intake, thereby reducing medical cost.

In a preferred embodiment, the amino acid sequence of the phospholipase is as follows:

AYAQWVIILIHNVGQQNVKIKNLNASWGKLYADGDKDTEVPASKYEGMVIAPGDQVQINACGREDAAEGTTGTFDLVDPNDSDKQVRHISWDCPWGTKANSWVVGGSNSKWMIEYTGQNLDSGALGTITVNTLKIGN(SEQ ID NO.1)。

in a preferred embodiment, the amino acid sequence of the linker peptide is as shown in SEQ ID NO.2 or SEQ ID NO. 3:

SGTPTPTPTPTGEF(SEQ ID NO.2)；

GGGSGGGSGGGS(SEQ ID NO.3)。

in a preferred embodiment, the amino acid sequence of the amphipathic protein is as set forth in SEQ ID No.4:

MKFFTAAALFAAVAIAGPVEVRTGGGSICPDGLFSNPQCCDTQLLGIIGLGCEVPSQTPRDGADFKNICAKTGDQALCCVLPIAGQDLLCQAAVGAA(SEQ ID NO.4)。

it should be noted that, in the present invention, the phospholipase sequence is SEQ ID No.1, the linker peptide sequence is SEQ ID No.2 or SEQ ID No.3, and the amphiphilic protein sequence is SEQ ID No.4, and the fusion protein composed of the phospholipase, the optional linker peptide, and the optional amphiphilic protein is an embodiment of the present invention, and the linker peptide plays a linking role between the phospholipase and the amphiphilic protein, and the specific sequence of the three is not particularly limited, for example, the amino acid sequence of the fusion protein from N-terminal to C-terminal may be phospholipase-linker peptide-amphiphilic protein, or may be amphiphilic protein-linker peptide-phospholipase.

Specific examples thereof include:

or the amphipathic protein shown in SEQ ID NO.4, the phospholipase shown in SEQ ID NO.1 and the connecting peptide shown in SEQ ID NO. 3.

In a preferred embodiment, the amino acid sequence of the fusion protein is:

or the like, or, alternatively,

the invention also protects the fusion protein-related biological material, and the biological material is any one of the following materials:

(a) A nucleic acid molecule encoding a fusion protein;

(b) An expression cassette comprising the nucleic acid molecule of (a);

The invention also provides a preparation method of the fusion protein, which comprises the steps of introducing the nucleic acid molecule for encoding the fusion protein into a host to obtain a recombinant expression system, and expressing the recombinant expression system to obtain the fusion protein.

In a preferred embodiment, the constructed nucleic acid molecule for encoding the fusion protein is enzymatically linked with an expression plasmid pPIC9 to obtain a recombinant expression vector, yeast is transfected, and the fusion protein is obtained by fermentation expression.

The invention also protects the application of the fusion protein in preparing the medicament for treating NASH.

A medicament for treating NASH, comprising the fusion protein of the invention and optional pharmaceutically acceptable auxiliary materials.

The treatment direction of the NASH treatment medicine provided by the invention is based on the original treatment direction of the medicine, and the administration times can be reduced, so that the compliance is improved. In particular, the medicament of the present invention may be administered in oral form. Although the dosage of the drug varies depending on the subject to be treated, the mode of administration, the symptoms and other factors, the drug of the present invention is effective over a relatively wide dosage range. The actual dosage should be determined by a physician in the light of the relevant circumstances, including the physical condition of the subject, the route of administration, the age, weight, individual response of the patient to the drug, the severity of the patient's symptoms, and the like, and therefore the above dosage range is not intended to limit the scope of the present invention in any way. Preferably, the effective amount of the fusion protein is 10-30 mg/person/day.

Regarding the choice of the excipients, those skilled in the art can select the excipients according to the conventional techniques and common general knowledge in the art, so as to obtain the drugs with different dosage forms, different administration modes and different states, and the excipients are not limited specifically herein.

In a preferred embodiment, the adjuvant comprises at least one of a water-soluble filler adjuvant, a pH adjuster, a stabilizer, water for injection, or an osmotic pressure regulator.

In a preferred embodiment, the water soluble filler excipient comprises at least one of mannitol, low molecular dextran, sorbitol, polyethylene glycol, glucose, lactose or galactose.

In a preferred embodiment, the pH adjusting agent comprises citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, potassium bicarbonate, or ammonium bicarbonate.

In a preferred embodiment, the stabilizer comprises at least one of EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, sodium dodecyl sulfate, or tris.

In a preferred embodiment, the stabilizer comprises at least one of sodium metabisulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000, tris.

In a preferred embodiment, the osmolality adjusting agent comprises sodium chloride and/or potassium chloride.

In a preferred embodiment, the administration of the NASH treatment drug is oral.

In a preferred embodiment, the dosage form of the medicament for treating NASH is lyophilized powder, tablets, capsules, oral liquid or granules.

A preparation method of a drug freeze-dried powder for treating NASH comprises the steps of uniformly mixing fusion protein and optional pharmaceutically acceptable auxiliary materials, and freeze-drying to obtain the drug freeze-dried powder for treating NASH.

In a preferred embodiment, a lyophilized pharmaceutical powder for treating NASH is prepared as follows:

taking a proper amount of fusion protein solution, adding a water-soluble filler auxiliary material, a stabilizer and an osmotic pressure regulator, adding a proper amount of water for injection, adjusting the pH value to 7 to dissolve the fusion protein solution, adding water to dilute the solution to a proper concentration, adding 0.1-0.5% of activated carbon, stirring the solution for 10-20 minutes at 0-10 ℃, removing the activated carbon, filtering and sterilizing the solution by adopting a microporous filter membrane, subpackaging the filtrate, preparing a white loose block by adopting a freeze-drying method, and sealing the block to obtain the freeze-dried powder injection.

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

Example 1: construction of the fusion protein expression vector of the present invention and purification of the fusion protein

The expression plasmid pPIC9 is subjected to double enzyme digestion, and then is connected with the constructed DNA fragment for encoding the fusion protein through T4 ligase. Once the expression vector encoding the complete fusion protein has been generated, it is used to transfect yeast to produce the fusion protein. The techniques for transforming or transfecting cells with the recombinant DNA vector can be those conventionally used in the art. The constructed plasmid is transformed into yeast by a heat shock transformation method, which is a conventional technique in the field. After the fusion protein is expressed for 16 hours, centrifuging the bacterial liquid, collecting, and ultrasonically crushing the thalli, wherein the operation procedure is as follows: 10cycles,15 sec/cycle. And (4) centrifuging the crushed bacterial liquid at a high speed, and injecting the supernatant into a hydrophobic chromatographic column for coarse purification. The eluted proteins were collected and dialyzed and concentrated using a centrifuge tube with a 10kDa semipermeable membrane. Separating the reaction mixture by using an RP-HPLC column, and simultaneously purifying the target fusion protein. The purified fusion protein was subjected to protein silver staining assay (SEQ ID NO. 5) (FIG. 1) and HPLC purity assay (SEQ ID NO. 6) (FIG. 2), and the purity of the fusion protein (SEQ ID NO. 6) was 96.8%.

Example 2:

10mg of the fusion protein of example 1, 0.2g of arginine as a stabilizer, and 5363 g of polyethylene glycol 60000.05g were placed in a container, 80ml of water for injection was added, stirred to dissolve, 8g of mannitol and 2g of sorbitol were added, stirred to dissolve, the pH was adjusted to 6.0-8.0 with 0.1mol/L sodium hydroxide, and water was added to 100ml. Adding 0.3g of active carbon, stirring at 10-25 deg.C for 20 min, decarbonizing, filtering with microporous membrane for sterilization, and packaging the filtrate at a volume of 1ml per bottle. Pre-freezing for 2 hr, drying under reduced pressure for 18 hr until the temperature of the sample reaches 15-20 deg.C, drying for 5 hr to obtain white loose block, and sealing to obtain white lyophilized powder for injection with specification of 100 μ g/piece.

Example 3

Taking 25mg of the fusion protein in the example 1, adding 0.05g of stabilizer dipotassium hydrogen phosphate and 0.9g of sodium chloride, putting the fusion protein into a container, adding 70ml of water for injection, stirring the mixture to dissolve the fusion protein, adding 12g of mannitol and 7g of lactose, stirring the mixture to dissolve the mixture, adjusting the pH value to 6.5-8.5 by using 0.1mol/L of sodium hydroxide, adding water to 100ml, adding 0.25g of activated carbon, stirring the mixture at 10-20 ℃ for 20 minutes, decarburizing the mixture, filtering the mixture by using a microporous filter membrane for sterilization, subpackaging the filtrate by 2ml per branch, pre-freezing the filtrate for 2 hours, drying the mixture under reduced pressure for 15 hours under refrigeration until the temperature of the sample reaches 15-20 ℃, drying the mixture for 5 hours again to prepare a white loose block, sealing the sample to obtain a white freeze-dried powder injection with the specification of 500 mu g/branch.

Example 4

Taking 50mg of the fusion protein in the example 1, adding 0.2g of stabilizer tris (hydroxymethyl) aminomethane, 0.1g of sodium metabisulfite and 0.9g of sodium chloride into a container, adding 80ml of water for injection, stirring and dissolving, adding sodium bicarbonate to adjust the pH to be 6-8, adding water to 100ml, adding 0.2g of activated carbon, stirring and adsorbing at 10-25 ℃ for 20 minutes, removing carbon, filtering and sterilizing by adopting a microporous filter membrane, and filling and sealing 2ml of each fusion protein to obtain a fusion protein liquid medicine with the specification of 1000 mu g/piece.

Example 5

5mg of the fusion protein of example 1, 0.01g of glutamic acid as a stabilizer, and 0.09g of potassium chloride were put in a vessel, 70ml of water for injection was added thereto and dissolved by stirring, and 2g of glucose and 1g of galactose were added thereto and dissolved by stirring. Adjusting pH to 7.0-8.5 with sodium hydroxide, adding water to 100ml, adding 0.05g of activated carbon, stirring and adsorbing at 10-25 deg.C for 20 min, removing carbon, filtering with microporous membrane for sterilization, and bottling with 1ml per bag to obtain fusion protein liquid medicine with specification of 50 μ g/bag.

Example 6 upregulation of the fusion protein (SEQ ID NO. 5) mitochondrial cell MFN2 protein

HIF-1B adipocytes were cultured according to the conventional cell biology and then co-incubated with the fusion protein (SEQ ID NO. 5) for 6, 12 hours. After the experiment is finished, the MFN2 and the phosphorylation level thereof are detected by a western-blotting method (FIG. 3). The upregulation of MFN2 and its phosphorylation level is closely related to adipocyte metabolism, and the goal of reducing triglyceride and cholesterol is achieved by adjusting the adipocyte metabolism, and it can be seen from the figure that the fusion protein (SEQ ID NO. 5) obviously activates the expression amount and phosphorylation level of MFN2 protein.

Example 7 fusion protein (SEQ ID NO. 6) having the function of treating NASH

HFD + CCL4 model animals were tested and the fusion protein (SEQ ID NO. 6) drug was administered orally 1 time/day. The biochemical markers, including triglyceride, total cholesterol and liver glycogen levels, were analyzed in the experimental animals 4 weeks after administration to the mice and 12 weeks after administration to the rats (table 1):

TABLE 1 therapeutic Effect of the fusion protein (SEQ ID NO. 6) on NASH model mice and rats

While particular embodiments of the present invention have been illustrated and described, it would be obvious that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

SEQUENCE LISTING

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Ala Tyr Ala Gln Trp Val Ile Ile Leu Ile His Asn Val Gly Gln Gln

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Asp Gly Asp Lys Asp Thr Glu Val Pro Ala Ser Lys Tyr Glu Gly Met

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Val Ile Ala Pro Gly Asp Gln Val Gln Ile Asn Ala Cys Gly Arg Glu

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Asp Ala Ala Glu Gly Thr Thr Gly Thr Phe Asp Leu Val Asp Pro Asn

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Asp Ser Asp Lys Gln Val Arg His Ile Ser Trp Asp Cys Pro Trp Gly

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130 135 140

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145 150 155 160

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165 170 175

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210 215 220

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Thr Leu Lys Ile Gly Asn

245

Claims

1. The application of fusion protein containing phospholipase in preparing a medicament for treating non-alcoholic steatohepatitis is disclosed, wherein the amino acid sequence of the fusion protein is as follows:

AYAQWVIILIHNVGQQNVKIKNLNASWGKLYADGDKDTEVPASKYEGMVIAPGDQVQINACGREDAAEGTTGTFDLVDPNDSDKQVRHISWDCPWGTKANSWVVGGSNSKWMIEYTGQNLDSGALGTITVNTLKIGNSGTPTPTPTPTGEFMKFFTAAALFAAVAIAGPVEVRTGGGSICPDGLFSNPQCCDTQLLGIIGLGCEVPSQTPRDGADFKNICAKTGDQALCCVLPIAGQDLLCQAAVGAA（SEQ ID NO.5）；

or the like, or, alternatively,

MKFFTAAALFAAVAIAGPVEVRTGGGSICPDGLFSNPQCCDTQLLGIIGLGCEVPSQTPRDGADFKNICAKTGDQALCCVLPIAGQDLLCQAAVGAAGGGSGGGSGGGSAYAQWVIILIHNVGQQNVKIKNLNASWGKLYADGDKDTEVPASKYEGMVIAPGDQVQINACGREDAAEGTTGTFDLVDPNDSDKQVRHISWDCPWGTKANSWVVGGSNSKWMIEYTGQNLDSGALGTITVNTLKIGN（SEQ ID NO.6）。

2. use of a fusion protein-related biomaterial according to claim 1 for the preparation of a medicament for the treatment of non-alcoholic steatohepatitis, wherein the biomaterial is any one of the following:

(a) A nucleic acid molecule encoding the fusion protein of claim 1;

(b) An expression cassette comprising the nucleic acid molecule of (a);

3. A medicament for treating non-alcoholic steatohepatitis, comprising the fusion protein of claim 1 and a pharmaceutically acceptable excipient.

4. The drug for treating nonalcoholic steatohepatitis as set forth in claim 3, wherein the effective amount of the fusion protein is 10-30 mg/person/day.

5. The drug for treating nonalcoholic steatohepatitis as set forth in claim 3, wherein the excipient comprises at least one of a water-soluble filler excipient, a pH regulator, a stabilizer, water for injection, or an osmotic pressure regulator.

6. The drug for treating nonalcoholic steatohepatitis as in claim 5, wherein the water-soluble filler excipient comprises at least one of mannitol, low-molecular dextran, sorbitol, polyethylene glycol, glucose, lactose or galactose.

7. The drug for treating nonalcoholic steatohepatitis according to claim 5, wherein the pH regulator comprises citric acid, phosphoric acid, lactic acid, tartaric acid, hydrochloric acid, potassium hydroxide, sodium hydroxide, ammonium hydroxide, sodium carbonate, potassium carbonate, ammonium carbonate, sodium bicarbonate, potassium bicarbonate, or ammonium bicarbonate.

8. The agent for treating nonalcoholic steatohepatitis of claim 5, wherein the stabilizer comprises at least one of EDTA-2Na, sodium thiosulfate, sodium metabisulfite, sodium sulfite, dipotassium hydrogen phosphate, sodium bicarbonate, sodium carbonate, arginine, glutamic acid, polyethylene glycol 6000, polyethylene glycol 4000, sodium lauryl sulfate, or tris (hydroxymethyl) aminomethane.

9. The agent of claim 8, wherein the stabilizer comprises at least one of sodium metabisulfite, dipotassium hydrogen phosphate, arginine, polyethylene glycol 6000, and tris (hydroxymethyl) aminomethane.

10. The agent for treating non-alcoholic steatohepatitis of claim 5, wherein the osmotic pressure regulator comprises sodium chloride and/or potassium chloride.

11. The drug for treating nonalcoholic steatohepatitis of claim 3, wherein the drug for treating nonalcoholic steatohepatitis is administered orally.

12. The drug for treating nonalcoholic steatohepatitis according to claim 3, wherein the dosage form of the drug for treating nonalcoholic steatohepatitis is freeze-dried powder, tablets, capsules, oral liquid or granules.

13. A preparation method of freeze-dried powder of a drug for treating non-alcoholic steatohepatitis is characterized in that the freeze-dried powder of the drug for treating non-alcoholic steatohepatitis is obtained by uniformly mixing the fusion protein in claim 1 with pharmaceutically acceptable auxiliary materials and then carrying out freeze-drying.