WO2011069408A1 - Polypeptide immunogen, preparation method and use thereof - Google Patents

Abstract

The invention discloses a polypeptide immunogen and a preparation method thereof. The polypeptide immunogen contains one polypeptide sequence which contains amino acid sequence 1, amino acid sequence 2, amino acid sequence 3 and amino acid sequence 4, wherein the amino acid sequences are connected with each other by connecting peptides consisting of a plurality of amino acid residues or are connected with each other covalently directly. The invention also provides the use of the polypeptide immunogen in preparation of vaccines or medicaments for treating HBV chronic infection persistent state and the related diseases such as secondary liver cirrhosis, liver cancer and the like.

Description

Polypeptide immunogen and preparation method and application thereof

Technical field

The invention relates to a polypeptide immunogen and a preparation method thereof, and the application of the polypeptide immunogen in preparing a vaccine or a medicament for treating a chronic infection status of HBV and related diseases such as secondary liver cirrhosis and liver cancer, belonging to a vaccine or a medicament Medical technology field.

Background technique

Hepatitis B is one of the infectious diseases that endanger the health of all human beings. Global hepatitis B virus infection accounts for about 30% of the population, and chronic hepatitis patients about 350 million. China is a highly endemic area. Hepatitis B virus carriers account for about 10% of the population (130 million), and chronic hepatitis patients are about 30 million. If they are unhealed, they can develop into cirrhosis and liver cancer.

At present, the main method for controlling the prevalence of hepatitis B is injection of preventive hepatitis B vaccine, but it is not effective for people infected with hepatitis B virus, and the vaccination rate is not very high for various reasons. Therefore, for a long period of time, hepatitis B will still be It is one of the important diseases that endanger human health.

It has been confirmed that HBV as a hepadnavirus does not directly cause liver cell damage, and the pathological and clinical consequences of its infection depend on the immune mechanism.

As an intracellular infection, the state of chronic persistent infection is mainly related to the weak cellular immune response. Among them, HBV-specific cytotoxic T lymphocytes (cytotoxic T The lymphocyte, CTL) response determines the final outcome of HBV infection. Infected with HBV after infection with HBV, the virus is cleared and cured; those with low CTL activity or undetectable develop chronically persistent infection and further develop into cirrhosis or liver cancer.

Therefore, breaking the immune tolerance status of patients with persistent HBV infection, and initiating HBV-specific CTL response in vivo, it can treat the chronic infection status of HBV and prevent related diseases such as secondary cirrhosis and liver cancer.

A research institution in the United States applied for four US patents on the basis of its own research. The main content of these patents relates to the CTL epitopes determined on the HBV antigen. These epitopes are derived from core antigen, surface antigen, polymerase and X antigen, respectively. These sites or structures are the sites recognized by the CTL induced by HBV. Chinese researcher Wen Yumei found that the antigen-antibody immunogenic complex can effectively eliminate viruses and disease antigens in some ducks, and thus it is possible to develop into a therapeutic vaccine for human use.

Currently, there is no specific treatment for chronic persistent infection of HBV (including viral carriers, chronic persistent hepatitis, and chronic active hepatitis). Interferon and lamivudine have specific therapeutic effects but do not eliminate the virus. Therefore, there is an urgent need to find new ways to effectively treat the chronic persistent infection status of HBV.

Summary of the invention

It is an object of the present invention to provide a polypeptide immunogen and a process for the preparation thereof.

Another object of the present invention is to provide use of the polypeptide immunogen for the preparation of a vaccine or medicament for the treatment of a chronic infection of HBV and its associated secondary cirrhosis, liver cancer and the like.

The polypeptide immunogen of the present invention comprises a polypeptide sequence comprising amino acid sequence 1, amino acid sequence 2, amino acid sequence 3 and amino acid sequence 4, wherein a linker peptide consisting of several amino acid residues between each amino acid sequence Segmental linkage or direct covalent linkage; the amino acid sequence 1 is a mixed sequence of a Th cell epitope sequence or a Th cell epitope sequence covalently linked to a basic region sequence of an HIV-1 source-derived TAT protein; The amino acid sequence 2 is the basic region sequence of the HIV-1 source-derived TAT protein; the amino acid sequence 3 is a hepatitis C virus-derived CTL epitope sequence or a hepatitis B virus-derived CTL epitope sequence and A mixed sequence consisting of a covalently linked sequence of a basic region of an HIV-1 derived TAT protein; the amino acid sequence 4 is a B cell epitope sequence derived from hepatitis B virus or a B cell derived from hepatitis B virus The sequence is covalently linked to the sequence of the basic region of the HIV-1 source-derived TAT protein.

The order of arrangement of the polypeptide sequences of the present invention from the N-terminus to the C-terminus may be any combination of amino acid sequence 1, amino acid sequence 2, amino acid sequence 3, and amino acid sequence 4. The preferred arrangement order is:

Amino acid sequence 1-amino acid sequence 2-amino acid sequence 3-amino acid sequence 4,

Amino acid sequence 1-amino acid sequence 3-amino acid sequence 4-amino acid sequence 2

Amino acid sequence 1-amino acid sequence 4-amino acid sequence 2-amino acid sequence 3,

Amino acid sequence 2-amino acid sequence 1-amino acid sequence 3-amino acid sequence 4,

And amino acid sequence 1-amino acid sequence 3-amino acid sequence 4,

Wherein: when amino acid sequence 2 is present, amino acid sequences 1, 3, 4 cannot be a mixed sequence;

When the arrangement order is amino acid sequence 1-amino acid sequence 3-amino acid sequence 4, amino acid sequence 1, amino acid sequence 3, and amino acid sequence 4 contain at least one mixed sequence.

The amino acid sequence 1 of the present invention is the 830-843 amino acid sequence QYIKANSKFIGITE or the universal Th cell epitope PADRE or the mixed sequence RKKRRQRRRQYIKANSKFIGITE or the mixed sequence RKKRRQRRRPADRE on the Th cell epitope derived from tetanus toxoid; the amino acid sequence 2 is HIV-1 The 48th-60th amino acid sequence GRKKRRQRRRPPQ or the 49th-57 amino acid sequence RKKRRQRRR or the 48th-57th amino acid sequence GRKKRRQRRR in the TAT protein; the amino acid sequence 3 is the 18th to 27th amino acid sequence FLPSDFFPSV or the mixed sequence RKKRRQRRRFLPSDFFPSV on the HBV core antigen The amino acid sequence 4 is HBV Pre The 14th amino acid sequence DPRVRGLYFPAGG or the 14-30 amino acid sequence DPRVRGLYFPAGGVSFG or the mixed sequence RKKRRQRRRDPRVRGLYFPAGGVSFG on the S2-derived B cell epitope.

The linking peptide between each amino acid sequence of the present invention is AAA, GGG or SSS; the polypeptide immunogen has a N-terminal amino acid sequence linked to a modified linking peptide KSS; the modification The palmitoyl group is covalently attached to the α or ε amino group of the N-terminal lysine residue of the peptide KSS, respectively or simultaneously.

The polypeptide immunogen of the present invention may have a plurality of polypeptide sequences, wherein the preferred polypeptide sequence is: CH3(CH2)14COKSSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (polypeptide 1),

CH3(CH2)14COK(CH3(CH2)14CO)SSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (polypeptide 2),

CH3(CH2)14COKSSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (polypeptide 3),

CH3(CH2)14COK(CH3(CH2)14CO)SSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (polypeptide 4),

CH3(CH2)14COKSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ (peptide 5),

CH3(CH2)14COK(CH3(CH2)14CO)SSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ (polypeptide 6),

CH3(CH2)14COKSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ (peptide 7),

CH3(CH2)14COK(CH3(CH2)14CO)SSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ (polypeptide 8),

CH3(CH2)14COKSSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (peptide 9),

CH3(CH2)14COK(CH3(CH2)14CO)SSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (peptide 10),

CH3(CH2)14COKSSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (peptide 11),

CH3(CH2)14COK(CH3(CH2)14CO)SSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (peptide 12),

CH3(CH2)14COKSSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG (peptide 13)

CH3(CH2)14COK(CH3(CH2)14CO)SSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAG

GVSFG (polypeptide 14),

CH3(CH2)14COKSSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV (polypeptide 15) or

CH3(CH2)14COK(CH3(CH2)14CO)SSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV (polypeptide 16).

A method for preparing a polypeptide immunogen, comprising the steps of:

a polypeptide solid phase synthesis of the polypeptide immunogen-resin, the polypeptide immunogen-resin representing a polypeptide immunogen bound to a resin;

b. cleavage of the polypeptide immunogen-resin to obtain a lysate;

c. The lysate is subjected to preliminary separation and purification by size exclusion chromatography, and then purified by reverse phase chromatography to obtain a polypeptide immunogen.

The use of a polypeptide immunogen for the preparation of a vaccine or medicament for the treatment of a persistent state of HBV chronic infection and its associated secondary cirrhosis, liver cancer disease.

The chronic infection status of HBV is chronic hepatitis B or hepatitis B virus carriers.

The vaccine or medicament also contains pharmaceutically acceptable excipients, adjuvants and carriers.

The vaccine or drug is administered by injection.

The English abbreviations of the commonly used amino acids of the present invention mean

A Ala alanine

D Asp aspartate

E Glu glutamic acid

F Phe phenylalanine

G Gly glycine

I Ile isoleucine

K Lys Lysine

L Leu leucine

N Asn asparagine

P Pro proline

Q Gln glutamate

R Arg arginine

S Ser serine

T Thr Threonine

V Val proline

Y Tyr tyrosine

The invention adopts solid phase chemical synthesis method to prepare the immunogen, and determines the optimal temperature of the reaction system, the feed ratio, the peptide resin cracking method and time, the purification conditions and the like.

In the method of the present invention, when the reaction temperature is 30-40 ° C, the HOBT/DCC coupling strategy is adopted, and the amino acid sequence is continuously synthesized, and the optimum molar ratio of the raw material and the resin is 3:1, and the average molar ratio of each amino acid or component is The coupling efficiency is over 99.5%; the peptide resin is cleaved by trifluoroacetic acid method, and the optimal lysis time is 90 minutes.

Purification is carried out in two steps: (1) preliminary purification by size exclusion chromatography, dimethyl sulfoxide (DMSO) as mobile phase, optimal column temperature 20-40 ° C; (2) reversed phase chromatography Highly purified, the best loading conditions, the best chromatographic packing, the best column temperature and other factors were studied. In the process of the invention, the optimal chromatography material is POROS 50 R1, the optimum column temperature is 22-34 °C.

Comparing the ethanol solution dosage form, the liposome suspension liquid dosage form, and the liposome lyophilized dosage form, it was proved that the liposome dosage form was superior to the ethanol solution dosage form in inducing Th1 polarization and inducing CTL activity, and the pain of subcutaneous injection was significantly reduced; Liposomal lyophilized formulations are significantly superior in stability to liposomal suspension formulations.

The present invention sets the optimal formulation of the liposome injection of the polypeptide immunogen. Among them, the above polypeptide immunogen, soybean phospholipid, palmitic acid, vitamin E and cholesterol are contained.

The liposome lyophilized dosage form of the present invention contains human albumin, mannitol and phosphate in addition to the above-mentioned polypeptide immunogen, phospholipid, cholesterol, palmitic acid and vitamin E.

The polypeptide immunogen of the present invention and its dosage form may be any known immunological means such as subcutaneous injection, intradermal injection, intraperitoneal injection, intravenous injection or the like. The immunization dose may be from 0.01 nmol to 20 nmol, and no additional adjuvant may be used. Immunization of HBV transgenic mice stimulates lymphocyte activation and proliferation, Th1 polarization and CTL responses, and inhibits HBV replication, HBV in blood, in a dose-dependent manner The DNA copy number decreased, and the HbsAg and HbeAg titers decreased or disappeared. This potency is not achievable with single-epitope polypeptides or multi-epitope polypeptides alone.

The present invention determines a polypeptide immunogen having a specific skeleton structure based on a hepatitis B epitope map. The polypeptide immunogen is effective in inducing Th1 polarization, HBV-specific CTL response, and inhibition of HBV replication in HBV transgenic mice. The experimental results indicate that the above-mentioned polypeptide immunogen can be developed into a novel therapeutic vaccine or drug for hepatitis B and its secondary cirrhosis and liver cancer.

detailed description

The following examples are merely illustrative of the invention, but are not intended to limit the invention.

Example 1

Peptide immunogen synthesis

The Fmoc solid phase chemical synthesis method was employed. The carboxy terminus is fixed, and the peptide chain is extended from the C-terminus to the N-terminus, and is carried out on a synthesizer. Fmoc amino acid was used as raw material, the sample loading was 0.75mmol, side chain protected amino acids Ser(tBU), Thr(tBU), Tyr(tBU), Gln(Trt), Asp(OtBU), Glu(OtBU), Arg(pbf) Asn (Trt), Lys (Boc), Wang-resin is selected as the solid phase carrier, the loading amount is 0.25mmol, the molar ratio of raw material to resin is 3:1, and the first amino acid coupled to the resin is DIC/HOBT. Method coupling, activation of the remaining amino acids and palmitic acid and their coupling using the HOBT/DCC method.

Example 2

Peptide immunogen cleavage

The use of TFA lysate (0.75 g phenol, 0.25 ml ethanedithiol, 0.5 ml thioanisole, 0.5 ml deionized water, 10.0 ml TFA) can minimize the occurrence of side reactions. The reaction concentration was 40 mg/ml (40 mg refers to the mass of the polypeptide immunogen-resin synthesized in Example 1, and ml refers to the volume of the lysate), the reaction temperature was 25 ° C, and the reaction time was 1.5 hours. The polypeptide molecule is cleaved from the resin, precipitated with diethyl ether, and rotary evaporated to give the crude peptide product.

Example 3

Preliminary purification of peptide immunogen

The crude peptide product was subjected to SEC loading of 55.0 ml to carry out preliminary purification of the polypeptide molecule. Chromatographic conditions: chromatography system: P-6000 pump and AKTAeplorer 100; chromatography column: diameter 25 mm, column length 850 mm; filler Sephadex LH20; mobile phase: DMSO; flow rate: 2.0 ml/min. Good purification results were obtained.

Example 4

Peptide immunogen purification method

Using column APS 50/275 POROS 50 R1 was prepared in batches. After the peptide resin was subjected to cleavage and preliminary purification, the sample was collected and divided into 7 times for high purification. Chromatographic conditions: chromatography system: AKTAexplorer 100; sample concentration: 10.52 mg / ml; sample loading: 11.0 ml; chromatography column: diameter 50 mm, column length 275 mm; filler POROS 50 R1; column temperature 34 ° C; mobile phase A: 30% ethanol - 10 mmol / L phosphoric acid; mobile phase B: 90% ethanol - 10 mmol / L phosphoric acid; gradient: 0-50% B, 5 CV; 50-100% B, 0.5 CV; 100-100% B, 0.5 CV; flow rate: 100.0 ml/min. The collected eluted peaks were mixed and stored at -20 ° C until use. The purity of this product is 98.5%, and the yield is 85%.

Example 5

Induction of Th1 polarization in HBV transgenic mice

HBV-DNA transgenic mice (ayw-type HBV whole gene (1.3 Kb) were used to infect Kunming mice). Animals were randomized into groups of 10, and polypeptide 5 was injected subcutaneously under the bilateral ribs and hind paws, and administered at 3 doses of 10, 100, and 1000 U/mouse, and boosted once a week for three times. IFN-α 2b (15000U/mouse) was used as a positive control drug, and physiological saline was used as a negative control drug. 10 days, 20 days, and 30 days after the end of the administration, the spleen of the mice was taken, and the spleen lymphocytes were isolated and stimulated with the test article 10 ng/ml for 3 days. The supernatant was taken and detected by ELISA. The secretion of cytokines such as IFN-γ and IL-4 in the Qing dynasty was analyzed, and the effect of the test drug on the Th1/Th2 type polarization of T cells was analyzed. As a result, strong IFN-γ secretion was detected, and no significant dose-effect relationship was observed in the detection of IL-4.

Example 6

Induction of CTL activity in HBV transgenic mice

HBV-DNA transgenic mice (ayw-type HBV whole gene (1.3 Kb) were used to infect Kunming mice). Animals were randomized into groups of 10, and polypeptide 5 was injected subcutaneously under the bilateral ribs and hind paws, and administered at 3 doses of 10, 100, and 1000 U/mouse, and boosted once a week for three times. IFN-α 2b (15000U/mouse) was used as a positive control drug, and physiological saline was used as a negative control drug. The spleen of the mice was taken before, and 10, 20, and 30 days after the end of the last administration. The spleen lymphocytes were isolated and stimulated with the test article 10 ng/ml for 3 days, and the IFN-γ secretion was detected by ELI-SPOT method. The frequency of expression of cells in peripheral blood lymphocytes. The results showed that on the 30th day after the end of immunization, the frequency of expression of IFN-γ secreting cells in peripheral blood lymphocytes increased with the increase of immunization dose, and the immune dose of 100 and 1000 U/mouse induced peripheral blood lymphocytes in vivo. The frequency of expression of IFN-γ secreting cells in cells increased significantly, and the highest detectable 3600±IFN-γ secreting cells/106 PBMC.

Example 7

Inhibition of viral surface antigen (HbsAg) in HBV transgenic mice

HBV-DNA transgenic mice (ayw-type HBV whole gene (1.3 Kb) were used to infect Kunming mice). Animals were randomized into groups of 10, and polypeptide 5 was injected subcutaneously under the bilateral ribs and hind paws, and administered at 3 doses of 10, 100, and 1000 U/mouse, and boosted once a week for three times. IFN-α 2b (15000U/mouse) was used as a positive control drug, and physiological saline was used as a negative control drug. Blood was taken 10 days, 20 days, and 30 days after the end of the administration and the last administration, and the serum was separated, and the serum HbsAg was measured by ELISA. The results showed that serum HbsAg was significantly reduced in a dose- and time-dependent manner.

Example 8

Inhibition of viral replication in HBV transgenic mice

HBV-DNA transgenic mice (ayw-type HBV whole gene (1.3 Kb) were used to infect Kunming mice). Animals were randomly divided into groups of 10, and peptide 5 was injected subcutaneously under the bilateral ribs and hind paws, and administered at 3 doses of 10, 100, 1000 U/mouse, and boosted once a week for three times. IFN-α 2b (15000U/mouse) was used as a positive control drug, and physiological saline was used as a negative control drug. Blood was taken 10 days, 20 days, and 30 days after the end of the administration and the last administration. The serum was separated and the serum HBV was detected by quantitative PCR. DNA copy number. The results showed that serum HBV DNA copy number was significantly reduced in a dose- and time-dependent manner.

Claims (10)

A polypeptide immunogen, characterized in that the polypeptide immunogen comprises a polypeptide sequence, and the polypeptide sequence comprises amino acid sequence 1, amino acid sequence 2, amino acid sequence 3 And amino acid sequence 4, wherein a linker peptide consisting of several amino acid residues between each amino acid sequence is ligated or directly covalently linked; the amino acid sequence 1 is a Th cell epitope sequence or a Th cell epitope sequence and AIDS a mixed sequence consisting of a covalently linked sequence of a basic region of a viral HIV-1 derived TAT protein; said amino acid sequence 2 is a basic region sequence of an HIV-1 derived TAT protein; said amino acid sequence 3 is a mixed sequence consisting of a CTL epitope sequence derived from hepatitis B virus or a CTL epitope sequence derived from hepatitis B virus and a basic region sequence of an HIV-1 derived TAT protein; said amino acid sequence 4 It is a mixed sequence consisting of a B cell epitope sequence derived from hepatitis B virus or a B cell epitope sequence derived from hepatitis B virus and a basic region sequence of a TAT protein derived from HIV HIV-1. The polypeptide immunogen according to claim 1, wherein the order of the polypeptide sequences from the N-terminus to the C-terminus is: Amino acid sequence 1-amino acid sequence 2-amino acid sequence 3-amino acid sequence 4, Amino acid sequence 1-amino acid sequence 3-amino acid sequence 4-amino acid sequence 2 Amino acid sequence 1-amino acid sequence 4-amino acid sequence 2-amino acid sequence 3, Amino acid sequence 2-amino acid sequence 1-amino acid sequence 3-amino acid sequence 4, And amino acid sequence 1-amino acid sequence 3-amino acid sequence 4, Wherein: when amino acid sequence 2 is present, amino acid sequences 1, 3, 4 cannot be a mixed sequence; When the arrangement order is amino acid sequence 1-amino acid sequence 3-amino acid sequence 4, amino acid sequence 1, amino acid sequence 3, and amino acid sequence 4 contain at least one mixed sequence. The polypeptide immunogen according to claim 1 or 2, wherein the amino acid sequence 1 is the amino acid sequence 830-843 of the Th cell epitope derived from tetanus toxoid, QYIKANSK FIGITE or the universal Th cell epitope PADRE Or a mixed sequence RKKRRQRRRQYIKANSKFIGITE or a mixed sequence RKKRRQRRRPADRE; the amino acid sequence 2 is HIV-1 The 48th-60th amino acid sequence GRKKRRQRRRPPQ or the 49th-57 amino acid sequence RKKRRQRRR or the 48th-57th amino acid sequence GRKKRRQRRR in the TAT protein; the amino acid sequence 3 is the 18th to 27th amino acid sequence FLPSDFFPSV or the mixed sequence RKKRRQRRRFLPSDFFPSV on the HBV core antigen The amino acid sequence 4 is HBV Pre The 14th amino acid sequence DPRVRGLYFPAGG or the 14-30 amino acid sequence DPRVRGLYFPAGGVSFG or the mixed sequence RKKRRQRRRDPRVRGLYFPAGGVSFG on the S2-derived B cell epitope. The polypeptide immunogen according to claim 1 or 2, wherein the linking peptide between the respective amino acid sequences is AAA, GGG or SSS; and the N-terminal amino acid sequence of the polypeptide immunogen is on the N-terminus The modified linking peptide KSS is ligated; the palmitoyl group is covalently linked to the α or ε amino group of the N-terminal lysine residue of the modified linking peptide KSS, respectively or simultaneously. The polypeptide immunogen according to claim 1, wherein the polypeptide sequence is: CH3(CH2)14COKSSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG, CH3(CH2)14COK(CH3(CH2)14CO)SSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG, CH3(CH2)14COKSSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG, CH3(CH2)14COK(CH3(CH2)14CO)SSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG, CH3(CH2)14COKSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ, CH3(CH2)14COK(CH3(CH2)14CO)SSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ, CH3(CH2)14COKSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ, CH3(CH2)14COK(CH3(CH2)14CO)SSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ, CH3(CH2)14COKSSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG, CH3(CH2)14COK(CH3(CH2)14CO)SSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG, CH3(CH2)14COKSSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG, CH3(CH2)14COK(CH3(CH2)14CO)SSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG, CH3(CH2)14COKSSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG, CH3(CH2)14COK(CH3(CH2)14CO)SSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG, CH3(CH2)14COKSSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV or CH3(CH2)14COK(CH3(CH2)14CO)SSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV. A method for preparing a polypeptide immunogen according to claim 1, 2 or 5, comprising the steps of: a. peptide solid phase chemical synthesis of polypeptide immunogen-resin, b. Pyrolysis of the polypeptide immunogen-resin using a trifluoroacetic acid method to obtain a lysate, c. The lysate is separated and purified by size exclusion chromatography, and then purified by reverse phase chromatography to obtain a polypeptide immunogen. Use of a polypeptide immunogen according to claim 1, 2 or 5 in the manufacture of a vaccine or medicament for the treatment of a chronic persistent infection state of HBV and its associated secondary cirrhosis, liver cancer disease. The use of the polypeptide immunogen according to claim 7 for the preparation of a vaccine or medicament for the treatment of chronic persistent infection of HBV and its associated secondary cirrhosis, liver cancer disease, characterized in that said HBV chronic infection persists It is a carrier of chronic hepatitis B or hepatitis B virus. The use of the polypeptide immunogen according to claim 7 for the preparation of a vaccine or medicament for the treatment of a chronic persistent infection state of HBV and its associated secondary cirrhosis, liver cancer disease, characterized in that said vaccine or medicament Also included are pharmaceutically acceptable adjuvants, adjuvants, and carriers. Use of the polypeptide immunogen according to claim 7 for the preparation of a vaccine or medicament for the treatment of a chronic persistent infection state of HBV and its associated secondary cirrhosis, liver cancer disease, characterized in that said vaccine or medicament The mode of administration is injection administration.