CN111072784B - Macromolecular furin inhibitor and preparation method and application thereof - Google Patents

Abstract

The invention discloses a macromolecular furin inhibitor, its preparation method and application, and belongs to the technical field of biomedicine. The macromolecular furin inhibitor is a macromolecular compound formed by covalently linking D-galactose, arginine-rich polypeptide and carrier protein, and its preparation method includes using a polypeptide synthesis method to synthesize arginine-rich polypeptide , galactose-linked arginine-enriched polypeptide, lactosylated arginine-enriched polypeptide linked to carrier protein and other steps. The macromolecular furin inhibitor of the present invention has high inhibition efficiency, strong ability to enter cells, low cytotoxicity and good liver targeting, so it has good potential for treating chronic hepatitis B.

Description

A kind of macromolecule furin inhibitor and its preparation method and application

technical field

The invention relates to the technical field of biomedicine, in particular to a macromolecular furin inhibitor and its preparation method and application.

Background technique

The proprotein convertase furin is a protease that exists in the trans-Golgi network of human or animal cells and can circulate in the cell membrane, endosome and Golgi. Physiologically, furin is involved in the synthesis of many growth factors, thus playing a key role in cell growth and differentiation; pathologically, furin is involved in processes such as tumorigenesis, viral infection, and bacterial toxin poisoning, indicating that furin plays an important role in various human diseases. A promising target for treatment.

Chronic HBV infection is the main infectious disease at present, it is easy to evolve into chronic hepatitis, cirrhosis and hepatocellular carcinoma, the prognosis is poor, and there is no radical cure. Chronic hepatitis B (CHB) is mainly divided into two types: early hepatitis B e antigen (HBeAg) positive CHB and late HBeAg negative CHB. Proprotein convertase furin is the key link in controlling the conversion of HBeAg precursor to HBeAg. In theory, inhibiting furin can achieve early treatment of HBeAg-positive CHB and prevention of late HBeAg-negative CHB by reducing HBeAg synthesis and increasing HBeAg precursor expression, thereby fundamentally improving the therapeutic effect of chronic hepatitis B and improving the prognosis of patients.

When hydrolyzing protein, the basic sequence of furin recognition site is N'-RXXR-C' (R is arginine, X is an indeterminate amino acid). At present, the methods of inhibiting furin mainly include gene silencing and small molecule inhibitors. Gene silencing is difficult to use in clinical treatment due to safety issues. Small molecule inhibitors are prepared based on the basic peptide sequence of the recognition site, such as decanoyl-RVKR-chloromethylketone (CMK), hexa-D-arginine (D6R), nena-D-arginine (D9R) and other arginine-rich set of compounds. These small molecule inhibitors have the following disadvantages:

1. Low efficiency: D6R and D9R often exhibit obvious inhibitory effects on furin in vitro, but their effects on cell models and animals are limited. The reason may be that furin mainly exists in cell organelles. Due to poor cell membrane permeability, concentrations of these inhibitors typically require hundreds of micromolar levels to produce an effect, making them extremely inefficient. At the same time, due to the poor permeability of the cell membrane, it stays on the cell membrane and damages the cell membrane. Therefore, there is basically no possibility of clinical application.

2. Easy to off-target: CMK is effective in vitro and in cell models, but it is easy to off-target. In addition to inhibiting furin, it also inhibits the trypsin-like activity of proteasome. For HBV infection, this off-target effect directly increases HBV replication, which is contrary to the purpose of treatment. In addition, the cytotoxicity is high. Therefore, CMK has basically no possibility of clinical application.

3. Large side effects: Furin is widely distributed in the body and has many functions. Inhibition of furin during the embryonic period directly leads to embryonic death. Non-selective inhibition of furin in late embryonic stages is not fatal, but may have unintended consequences. It has been reported that furin knockdown in mouse peripheral blood lymphocytes can lead to autoimmune responses.

In view of the above technical difficulties, there is an urgent need in this field for a novel artificially constructed furin inhibitor, in order to fundamentally improve the therapeutic effect of chronic hepatitis B and improve the prognosis of patients.

Contents of the invention

In order to overcome the deficiencies in the prior art, the object of the present invention is to provide a macromolecular furin inhibitor, which has high inhibition efficiency, low cytotoxicity, and good liver targeting, so it has a good therapeutic effect on chronic Potential for hepatitis B.

In order to solve the above problems, the technical scheme adopted in the present invention is as follows:

A macromolecular furin inhibitor, which is a macromolecular compound formed by covalently linking D-galactose, arginine-rich polypeptide and carrier protein.

As a preferred embodiment of the present invention, the sequence of the arginine-rich polypeptide is shown in SEQ ID No.1 or SEQ ID No.2 or the following general formula: RX ₁ X ₂ R, wherein X ₁ is Gla, Thr , Val or Arg, X ₂ is Lys or Arg.

As a preferred embodiment of the present invention, the carrier protein is bovine serum albumin or human serum albumin.

As a preferred embodiment of the present invention, the structure of the macromolecular furin inhibitor is as follows: D-galactose-TRRRRRRRRC-BSA.

As another preferred embodiment of the present invention, the structure of the macromolecular furin inhibitor is as follows: D-galactose-TRAKRRTKRTC-BSA.

As a preferred embodiment of the present invention, the molecular weight of the macromolecular furin inhibitor is 60-200 KDa.

In a second aspect of the present invention, a method for preparing the macromolecular furin inhibitor as described above is provided, comprising the following steps:

A. Synthesis of arginine-enriched polypeptides: Synthesize arginine-enriched polypeptides using a polypeptide synthesis method, and the synthesized crude product contains the peptides represented by SEQ ID No.1 or SEQ ID No.2 or the general formula RX ₁ X ₂ R The amino acid sequence shown; the crude product of the arginine-enriched polypeptide was purified by HPLC, and the purified solution was freeze-dried to obtain the pure product of the arginine-enriched polypeptide;

B. Galactose-linked arginine-enriched polypeptide: Dissolve the arginine-enriched polypeptide in MeOH solution, add galactose, NaBH ₃ CN and AcOH to react; cut the product in cutting solution to obtain lactosylated arginine The acid-enriched polypeptide crude product is purified by HPLC, and the purified solution is lyophilized to obtain the pure lactosylated arginine-enriched polypeptide;

C. Lactosylated arginine-enriched polypeptide connected to carrier protein: After dissolving maleimide, add carrier protein solution for activation, and lactosylated arginine-enriched polypeptide is dissolved in PBS solution and slowly dripped into the activated carrier The reaction is carried out in a protein solution; the reaction solution is dialyzed, and the dialysate is taken out and freeze-dried to obtain the macromolecular furin inhibitor.

In the third aspect of the present invention, the application of the macromolecular furin inhibitor as described above in the preparation of a drug for treating chronic hepatitis B is provided.

In the fourth aspect of the present invention, a pharmaceutical composition is provided, which contains the above-mentioned macromolecular furin inhibitor and a pharmaceutically acceptable carrier or excipient.

As a preferred embodiment of the present invention, the pharmaceutical composition is freeze-dried powder.

Compared with the prior art, the beneficial effects of the present invention are:

In the present invention, D-galactose-threonine is used as the first amino acid to synthesize polypeptides, and N-terminal lactosylated arginine-enriched peptides are prepared. SH is attached to the primary amino group of the carrier protein lysine, thus forming a macromolecular furin inhibitor. The macromolecule furin inhibitor obtained by the present invention can utilize the targeting of ASGP receptors on the liver cell membrane to well enter the main places such as the Golgi body where furin exists in the cell, and has good liver targeting, thereby greatly improving the furin resistance. Inhibition efficiency in vivo, and low cytotoxicity, effectively improves drug efficiency, and reduces side effects caused by off-target and pan-exposure, so the macromolecular furin inhibitor of the present invention has good potential for treating chronic hepatitis B. Drugs for the treatment of chronic hepatitis B provide new ideas.

Description of drawings

Fig. 1 is the structural representation of the macromolecule furin inhibitor of the present invention;

Fig. 2 is the gel electrophoresis band diagram of the macromolecule furin inhibitor of the present invention;

Fig. 3 is the comparison chart of the inhibition efficiency analysis of the macromolecular furin inhibitor of the present invention to HBeAg;

Fig. 4 is the analytical diagram of the influence of the macromolecular furin inhibitor of the present invention on the expression level of HBeAg precursor;

Fig. 5 is the cytotoxicity analysis chart of the macromolecule furin inhibitor of the present invention;

Fig. 6 is an analysis diagram of the cell-entry ability of the macromolecular furin inhibitor of the present invention;

Fig. 7 is a distribution diagram of the labeled polypeptide of the macromolecular furin inhibitor of the present invention in mice.

detailed description

The present invention will be described in further detail below in conjunction with the accompanying drawings and specific embodiments.

The structure of the macromolecular furin inhibitor of the present invention is shown in Figure 1, which is a novel macromolecular compound formed by covalently linking D-galactose, arginine-rich polypeptide and carrier protein. Wherein, the sequence of the arginine-rich polypeptide is shown in SEQ ID No.1 or SEQ ID No.2 or the following general formula: RX ₁ X ₂ R, wherein X ₁ is Gla, Thr, Val or Arg, X ₂ It is Lys or Arg; the carrier protein is bovine serum albumin or human serum albumin. The molecular weight of the macromolecule furin inhibitor is 60-200KDa.

When bovine serum albumin (BSA) is used as the carrier protein and the sequence shown in SEQ ID No.1 is an arginine-rich polypeptide, the structure of the macromolecular furin inhibitor is as follows: D-galactose-TRRRRRRRRC-BSA(gal-D9R -BSA). When bovine serum albumin (BSA) is used as the carrier protein and the sequence shown in SEQ ID No.2 is an arginine-rich polypeptide, the structure of the macromolecular furin inhibitor is as follows: D-galactose-TRAKRRTKRTC-BSA(gal-FPd -BSA).

1. The main physical and chemical properties of macromolecular furin inhibitors

Taking the carrier protein as BSA as an example, the molecular weight of the macromolecular furin inhibitor is about 100kDa, the molecular weight of lactosylated arginine-rich polypeptide (gal-FPd) is 1.54kDa, and the molecular weight of BSA is 66.4kDa, indicating that a BSA molecule binds About 20 molecules of lactosylated arginine-rich peptide (gal-FPd) were obtained.

Two, the preparation method of macromolecule furin inhibitor

The preparation method of macromolecule furin inhibitor comprises the following steps:

A. Synthetic arginine-enriched polypeptide:

The arginine-rich polypeptide is synthesized by a peptide synthesis method, and the synthesized crude product contains the amino acid sequence shown in SEQ ID No.1 or SEQ ID No.2 or the general formula RX ₁ X ₂ R, and the specific process is as follows:

①Resin swelling: put 2-Chlorotrityl Chloride Resin resin into the reaction tube, add DCM (15ml/g), shake for 30min;

②Connect Fmoc-D-Cys(Trt)-OH: filter the solvent through the sand core, add 3 times molar excess of the corresponding Fmoc-D-Cys(Trt)-OH, then add 10 times molar excess of DIEA, and finally Add DMF to dissolve, shake for 30 minutes, cap the head with methanol for 30 minutes;

③ Deprotection: remove the solvent, add 20% piperidine/DMF solution (15ml/g) for 5min, remove and add 20% piperidine/DMF solution (15ml/g) for 15min;

④ Detection: Take out the piperidine solution, take more than a dozen resins, wash them three times with ethanol, add one drop each of ninhydrin, KCN, and phenol solutions, heat at 105°C-110°C for 5 minutes, and turn dark blue as a positive reaction;

⑤Washing: DMF (10ml/g) twice, methanol (10ml/g) twice, DMF (10ml/g) twice;

⑥Condensation: Put in a three-fold excess of Fmoc-D-Arg(Pbf)-OH and a three-fold excess of HBTU, dissolve them with as little DMF as possible, add to the reaction tube, immediately add a ten-fold excess of DIEA, and react for 30 minutes;

⑦ Drying and washing: Dry the lysate with nitrogen as much as possible, wash with ether six times, and then evaporate to dry at room temperature to obtain the crude polypeptide;

⑧Purification: Purify the polypeptide by HPLC, and freeze-dry the purified solution to obtain pure arginine-enriched polypeptide;

⑨Identification: Take a small amount of finished peptides respectively, and do molecular weight identification by MS and purity identification by HPLC analysis;

⑩Preservation: Store the white powdered peptide in a sealed package at -20°C.

B. Galactose-linked arginine-enriched polypeptide:

①Connection: Dissolve the arginine-enriched polypeptide in 100ml MeOH solution, add galactose (1eq), NaBH ₃ CN (1.1eq) and AcOH (0.3eq), stir and react at 55°C overnight, and MS detects the reaction; reaction system Distillation under reduced pressure removes MeOH;

②Cutting: Prepare cutting fluid (10ml/g): TFA 94.5%; water 1%; EDT 2.5%; TIS 2.5%, cut the polypeptide in the cutting fluid for 120min;

③ Drying and washing: Dry the lysate with nitrogen as much as possible, wash it with ether six times, and then dry it at room temperature to obtain the crude lactosylated arginine-enriched polypeptide;

④Purification: Purify the polypeptide by HPLC, and freeze-dry the purified solution to obtain the pure lactosylated arginine-enriched polypeptide;

⑤Identification: Take a small amount of finished peptides for molecular weight identification by MS and purity identification by HPLC analysis;

⑥Preservation: Pack the peptide in white powder form in a sealed package and store it at -20°C.

C. Lactosylated arginine enriched polypeptide linked to carrier protein:

①Connection: Dissolve the carrier protein to an appropriate concentration, dissolve the maleimide (SMCC) with a small amount of DMF, add the carrier protein solution for activation for 2 hours, dissolve the lactosylated arginine-enriched polypeptide with PBS solution, and slowly drop into it Activated carrier protein solution, room temperature, magnetic stirring for 5h;

② Dialysis: Put the reaction solution in a dialysis bag, put it in PBS buffer solution and dialyze at 4°C for 48 hours, and replace the dialysis solution twice;

③Preservation: The dialysate was taken out and freeze-dried to obtain the macromolecular furin inhibitor. The product was subpackaged into 1mg/cartridge sealed packages and stored at -20°C.

3. Identification of macromolecular furin inhibitors

Take the lyophilized macromolecular furin inhibitor and dissolve it in 1ml PBS or first dissolve it in 0.1ml DMSO and then add 0.9ml PBS to a final concentration of 1μg/μl for use. Take 10 μl of the solution, use 8% polyacrylamide gel electrophoresis to separate, transfer to PDF membrane, use anti-human serum albumin polyclonal antibody and enhanced chemiluminescence reagent for immunospot detection, and obtain strips on a fluorescence/luminescence imager With images, the result is shown in Figure 2.

Based on the carrier protein with known molecular weight, the number of molecules linked to the lactosylated polypeptide by the carrier protein can be determined according to the increase in the molecular weight of the macromolecular furin inhibitor band. As shown in Figure 2, taking the carrier protein BSA as an example, the protein has a molecular weight of 66.4 kDa and about 59 lysines, among which there are about 30-35 primary amino groups available for connection. Taking gal-FPd-BSA in Figure 2 as an example, the average number of BSA-linked polypeptide molecules (~20)=[Inhibitor molecular weight (~100kDa)-BSA molecular weight (66.4kDa)]/polypeptide molecular weight (15.4kDa).

Method for determining the molecular weight of the linker: according to the mobility, compare the molecular marker, and calculate it by professional software. After repeated optimization, BSA connects 20 polypeptide molecules on average. An average of less than 15 is a failure of the preparation. It has been proved by many experiments that the connection efficiency of gal-FPd-BSA is generally higher than that of gal-D9R-BSA.

4. Performance verification of lactosylation macromolecule furin inhibitor

Since furin is involved in many physiological and pathological processes, the macromolecule furin inhibitor of the present invention has many potential uses. In this experiment, only macromolecular furin inhibitors take the effect on HBeAg synthesis as an example to explore the effects of macromolecular furin inhibitors on HBeAg inhibition efficiency and the expression of HBeAg precursors, as well as the cytotoxicity and liver targeting of macromolecular furin inhibitors. sex.

1. The effect of macromolecular furin inhibitors on the inhibitory efficiency of HBeAg

The experimental process is as follows: the HBV transformed model cell HepG2.2.15 and the infected model cell HepG2-NTCP were cultured in the modified Eagle's medium containing 10% fetal bovine serum, and were added with 380g/mL of G418 and 2g/mL of puromycin , change the medium every 24 hours, add macromolecular furin inhibitor and control inhibitor when 70% of cells grow continuously, collect cell supernatant after 48 hours, use commercial ELISA kit to detect HBsAg and HBeAg levels, the results are shown in Figure 3 .

It can be seen from Figure 3 that on the HepG2.2.15 cell model, 100 μM D6R and an equal dose of lactosylated arginine-rich polypeptide (gal-FPd) acted for 48 hours have similar HBeAg secretion inhibitory ability (Figure 3-A). However, 5 μM gal-FPd-BSA (which has a comparable number of gal-FPd molecules to 100 μM gal-FPd) increased its ability to inhibit HBeAg by 5 times (Fig. 3-B). There was a similar inhibitory effect on the HBV-infected HepG2-NTCP cell model (Fig. 3-C). In addition, macromolecular furin inhibitors have better drug withdrawal effects, suggesting that repeated administration may obtain better therapeutic effects in the future.

2. The effect of macromolecular furin inhibitors on the expression of HBeAg precursor

Taking the macromolecular furin inhibitor gal-FPd-BSA as an example, 1 μM gal-FPd-BSA was used for 48 hours on the HBV-infected HepG2-NTCP cell model, and the anti-HBc polyclonal antibody labeled with Cy3 was used for flow cytometry. The detection results are shown in Figure 4.

It can be seen from Figure 4 that the expression of HBeAg precursor on the cell membrane treated with gal-FPd-BSA was significantly increased.

3. Cytotoxicity of macromolecular furin inhibitors

Taking the macromolecular furin inhibitor gal-FPd-BSA as an example, on the HepG2.2.15 cell model, use 1-25 μM gal-FPd-BSA, and use 20-500 μM equivalent dose of gal-FPd as the control, act for 48 hours, and use MTT analysis 5 μM gal-FPd-BSA and 100 μM gal-FPd were used for research in HepG2-NTCP, and the results are shown in Figure 5.

It can be seen from Figure 5 that both gal-FPd and gal-FPd-BSA had no obvious toxicity to cells, but at a concentration equivalent to 500 μM gal-FPd, the toxicity of gal-FPd-BSA treated cells was significantly reduced (P<0.05) (Fig. 5-A). On the HBV-infected HepG2-NTCP cell model, gal-FPd-BSA also had no significant cytotoxicity (Fig. 5-B).

4. The ability of macromolecular furin inhibitors to enter cells

One of the purposes of developing lactosylated macromolecular furin inhibitors is to improve their ability to enter cells. In order to evaluate the ability of lactosylated macromolecular furin inhibitors to enter cells, taking the macromolecular furin inhibitor gal-FPd-BSA as an example, Cy3 Fluorescein-labeled gal-FPd-BSA and FPd-BSA were treated with HepG2.2.15 cells, and the confocal microscopy was used to detect the entry into the cytoplasm. The results are shown in Figure 6.

It can be seen from Figure 6 that gal-FPd-BSA can enter the cell, and FPd-BSA without galactose only stays on the cell membrane (Figure 6), which indicates that the macromolecular furin inhibitor can enter the cell efficiently, which may be its Basis for improved efficacy and reduced cytotoxicity.

5. Liver targeting of macromolecular furin inhibitors

Another important purpose of developing the lactosylated macromolecule furin inhibitor is to improve its organ targeting, so as to improve the therapeutic effect and reduce its systemic side effects, such as the autoimmune reaction caused by the inhibition of furin in peripheral blood lymphocytes.

Taking the macromolecular furin inhibitor gal-FPd-BSA as an example, the targeting evaluation was carried out in normal BALB/c mice. The specific process is as follows: 10 mice were taken each, and 5 μM Cy3 fluorescence-labeled gal-FPd-BSA and 100 μl of FPd-BSA were injected into the tail vein respectively. After 60 minutes, the mice were sacrificed by cervical dissection, and blood and tissues were collected. Accurately weigh 10 mg of each tissue, homogenate, collect the supernatant and blood and detect it on a fluorescence/luminescence detector, the wavelength of excitation light is 550nm, the wavelength of emission light is 600nm, and Cy3-labeled gal-FPd-BSA and Cy3 with different dilutions FPd-BSA makes a standard curve, and quantifies the content of Cy3-labeled gal-FPd-BSA and Cy3-labeled FPd-BSA in each organ, as the percentage of injected dose per gram of tissue or per milliliter of blood (%ID/g or %ID/ mL, ID is the injection dose) represents the relative content of each tissue for statistical analysis. The distribution of labeled polypeptides in each tissue is shown in Figure 7.

It can be seen from Figure 7 that, relative to FPd-BSA, gal-FPd-BSA increased in liver, spleen and lung tissues, and the liver tissue increased the most, which was 5 times that in blood. In contrast, FPd-BSA had significantly higher concentrations in blood than gal-FPd-BSA. The above results indicated that, compared with FPd-BSA, gal-FPd-BSA had better tissue affinity and obvious liver targeting.

From the above experiments, it can be seen that compared with the small molecule inhibitor D6R and the lactosylated arginine-enriched polypeptide, the lactosylated macromolecular furin inhibitor linked to the carrier protein of the present invention has the following advantages: the inhibitory efficiency to HBeAg is high and can significantly Increase the expression of HBeAg precursor, strong ability to enter cells, low cytotoxicity, and good liver targeting. The above advantages indicate that, compared with the current small molecule inhibitors, the lactosylated arginine-rich peptide macromolecule furin inhibitor has better therapeutic potential for chronic hepatitis B.

In summary, the macromolecular furin inhibitor of the present invention can utilize the ASGP receptor targeting on the liver cell membrane to well enter the main places such as the Golgi apparatus where furin exists in the cell, and has good liver targeting, thereby greatly improving The in vivo inhibition efficiency to furin is improved, and the cytotoxicity is low, the drug efficiency is effectively improved, and the side effects caused by off-target and pan-exposure are reduced, so the macromolecular furin inhibitor of the present invention has a good effect on treating chronic hepatitis B Potential, providing a new idea for the development of drugs for the treatment of chronic hepatitis B.

Therefore, the present invention also provides the application of the above-mentioned macromolecular furin inhibitor in the preparation of medicaments for treating chronic hepatitis B. Furthermore, the present invention also provides a pharmaceutical composition, which contains the aforementioned macromolecular furin inhibitor and a pharmaceutically acceptable carrier or excipient. Preferably, the pharmaceutical composition is a freeze-dried powder, and the freeze-dried powder is stable at -20°C. The freeze-dried powder gal-FPd-BSA is dissolved in normal saline and PBS for use, and the freeze-dried powder gal-D9R-BSA needs to be dissolved in DMSO before being dissolved in normal saline and PBS.

The above-mentioned embodiment is only a preferred embodiment of the present invention, and cannot be used to limit the protection scope of the present invention. Any insubstantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of the present invention. Scope of protection claimed.

SEQUENCE LISTING

<110> The Fifth Affiliated Hospital of Sun Yat-sen University

<120> A macromolecular furin inhibitor and its preparation method and application

<130> 2019

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 11

<212> PRT

<213> Artificial sequence

<400> 1

Thr Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Cys

1 5 10

<210> 2

<211> 11

<212> PRT

<213> Artificial sequence

<400> 2

Thr Arg Ala Lys Arg Arg Thr Lys Arg Thr Cys

1 5 10

Claims (6)

1. A macromolecular furin inhibitor, characterized in that: it is a macromolecular compound formed by covalently linking D-galactose, arginine-rich polypeptide and carrier protein; said macromolecular furin inhibitor The structure of is as follows: D-Galactose-TRAKRRTKRTC-BSA. 2. The macromolecular furin inhibitor according to claim 1, characterized in that: the molecular weight of the macromolecular furin inhibitor is 60-200KDa. 3. a preparation method of the macromolecule furin inhibitor as claimed in claim 1 or 2, is characterized in that: comprise the following steps: A. Synthesis of arginine-enriched polypeptide: arginine-enriched polypeptide is synthesized by a polypeptide synthesis method, and the synthesized crude product contains the amino acid sequence shown in SEQ ID No.2; the arginine-enriched polypeptide is purified by HPLC For the crude product, the purified solution is lyophilized to obtain the pure arginine-enriched polypeptide; B. Galactose-linked arginine-enriched polypeptide: Dissolve the arginine-enriched polypeptide in MeOH solution, add galactose, NaBH ₃ CN and AcOH to react; cut the product in cutting solution to obtain galactosylated essence The crude product of amino acid-enriched polypeptide is purified by HPLC, and the purified solution is lyophilized to obtain the pure product of galactosylated arginine-enriched polypeptide; C. Galactosylated arginine-enriched polypeptide connected to carrier protein: After dissolving maleimide, add carrier protein solution for activation, and galactosylated arginine-enriched polypeptide is dissolved in PBS solution and slowly dripped into it for activation. Carry out the reaction in the carrier protein solution; dialyze the reaction solution, take out the dialysate and freeze-dry to obtain the macromolecular furin inhibitor. 4. The application of the macromolecular furin inhibitor as claimed in claim 1 or 2 in the preparation of a medicament for the treatment of chronic hepatitis B. 5. A pharmaceutical composition, characterized in that it contains the macromolecular furin inhibitor as claimed in claim 1 or 2 and a pharmaceutically acceptable carrier or excipient. 6. The pharmaceutical composition according to claim 5, characterized in that: the pharmaceutical composition is freeze-dried powder.

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