CN101717433A - Polypeptide immunogen, preparation method and application thereof - Google Patents

Polypeptide immunogen, preparation method and application thereof Download PDF

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CN101717433A
CN101717433A CN200910227365A CN200910227365A CN101717433A CN 101717433 A CN101717433 A CN 101717433A CN 200910227365 A CN200910227365 A CN 200910227365A CN 200910227365 A CN200910227365 A CN 200910227365A CN 101717433 A CN101717433 A CN 101717433A
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aminoacid sequence
sequence
arg
gly
pro
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张嘎
张贲
韩斌
冯长访
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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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    • C12N2730/00011—Details
    • C12N2730/10011—Hepadnaviridae
    • C12N2730/10111—Orthohepadnavirus, e.g. hepatitis B virus
    • C12N2730/10134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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Abstract

The invention discloses a polypeptide immunogen and a preparation method thereof. The polypeptide immunogen contains one polypeptide sequence which contains an amino acid sequence 1, an amino acid sequence 2, an amino acid sequence 3 and an 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 application of the polypeptide immunogen in preparation of vaccines or medicaments for treating the diseases of HBV continuous state of chronic infection and related secondary biliary cirrhosis, liver cancer and the like.

Description

A kind of polypeptide immunogen and its production and application
Technical field
The present invention relates to a kind of polypeptide immunogen and preparation method thereof, and this polypeptide immunogen is used for the treatment of the vaccine of diseases such as HBV chronic infection persistent state and relevant Secondary cases liver cirrhosis thereof, liver cancer or the application in the medicine in preparation, belongs to medical technical field.
Background technology
Hepatitis B is one of communicable disease of harm universe health.The hepatitis b virus infected person in the whole world accounts for crowd's 30%, chronic hepatitis patient about 3.5 hundred million.China belongs to high popular district, and the carrier of hepatitis B virus accounts for 10% (1.3 hundred million) of population, and chronic hepatitis patient is about 0.3 hundred million, then can develop into liver cirrhosis, liver cancer as its protracted course of disease.
Control hepatitis B popular main method is the injection hepatitis B preventing vaccine at present, but it is invalid to the hepatitis B virus infection person, and rate of vaccination neither be very high because of various reasons, therefore, from now on quite over a long time in, hepatitis B will be one of important diseases of harm humans health.
Verified at present: HBV has a liking for hepatovirus as a kind of, can not directly cause hepatocellular injury, and the pathology and the clinical consequences of its infection depend on immunologic mechanism.
As infecting in the cell, chronic persistent infection state is mainly too weak relevant with the body cell immunne response.Wherein, (cytotoxic T lymphocyte, CTL) reaction has determined the net result that HBV infects to the HBV specificity cell toxicity T lymphocyte.Infecting behind the HBV the active high person's body inner virus of CTL is eliminated and fully recovers; The active the infected low or that do not detect of CTL then develops into chronic persistent infection state, and is further development of liver cirrhosis or liver cancer.
Therefore, break HBV persistent infection patient's immune tolerance state, start the reaction of HBV specific CTL in vivo, can treat HBV chronic infection persistent state and prevent diseases such as its relevant Secondary cases liver cirrhosis, liver cancer.
One tame research institution of the U.S. oneself research the basis on SEPARATE APPLICATION 4 United States Patent (USP)s.These patents to the effect that about determined CTL epi-position on HBV antigen.These epi-positions derive from cAg, surface antigen, polymerase and X antigen respectively.These positions or structure are the positions that the inductive CTL of HBV institute is discerned.China scientific research personnel hears beautiful plum and finds Ag-Ab immunogenic complex removing virus and disease antigen in the part duck effectively, thinks that thus it is possible developing into the therapeutic vaccine of using for human body.
Current, the chronic persistent infection state of HBV (comprising virus carrier, chronic persistent hepatitis and chronic active hepatitis) still there is not the specific treatment method.Though Interferon, rabbit and lamifudin have specific result of treatment, can not remove virus.Therefore, be badly in need of at present the novel method of seeking effectively to treat the chronic persistent infection state of HBV.
Summary of the invention
The object of the present invention is to provide a kind of polypeptide immunogen and preparation method thereof.
Another object of the present invention is to provide this polypeptide immunogen to be used for the treatment of the vaccine of diseases such as HBV chronic infection persistent state and relevant Secondary cases liver cirrhosis thereof, liver cancer or the application in the medicine in preparation.
Polypeptide immunogen of the present invention, contain a peptide sequence, this peptide sequence contains aminoacid sequence 1, aminoacid sequence 2, aminoacid sequence 3 and aminoacid sequence 4, and wherein, the joining peptide of being made up of several amino-acid residues between each aminoacid sequence connects or be directly covalently bound; Described aminoacid sequence 1 is the link to each other mixed sequence of formation of Th cell epitope sequence or Th cell epitope sequence and the proteic alkaline regional sequence covalency of TAT in virus of AIDS HIV-1 source; Described aminoacid sequence 2 is the proteic alkaline regional sequences of TAT in virus of AIDS HIV-1 source; Described aminoacid sequence 3 is link to each other with the proteic alkaline regional sequence covalency of TAT in virus of AIDS HIV-1 source mixed sequences of formation of the CTL epitope sequences in the CTL epitope sequences in hepatitis B virus source or hepatitis B virus source; Described aminoacid sequence 4 is link to each other with the proteic alkaline regional sequence covalency of TAT in virus of AIDS HIV-1 source mixed sequences of formation of the B cell epitope sequence in the B cell epitope sequence in hepatitis B virus source or hepatitis B virus source.
It can be the arbitrary combination of aminoacid sequence 1, aminoacid sequence 2, aminoacid sequence 3, aminoacid sequence 4 that peptide sequence of the present invention is held the ordering of C end from N, and wherein, preferred ordering is:
Aminoacid sequence 1-aminoacid sequence 2-aminoacid sequence 3-aminoacid sequence 4,
Aminoacid sequence 1-aminoacid sequence 3-aminoacid sequence 4-aminoacid sequence 2,
Aminoacid sequence 1-aminoacid sequence 4-aminoacid sequence 2-aminoacid sequence 3,
Aminoacid sequence 2-aminoacid sequence 1-aminoacid sequence 3-aminoacid sequence 4,
With aminoacid sequence 1-aminoacid sequence 3-aminoacid sequence 4,
Wherein: when aminoacid sequence 2 existed, aminoacid sequence 1,3,4 can not be mixed sequence;
When ordering is aminoacid sequence 1-aminoacid sequence 3-aminoacid sequence 4, contain a mixed sequence at least in aminoacid sequence 1, aminoacid sequence 3, the aminoacid sequence 4.
Aminoacid sequence 1 of the present invention is 830-843 aminoacid sequence QYIKANSKFIGITE or general Th cell epitope PADRE or mixed sequence RKKRRQRRRQYIKANSKFIGITE or the mixed sequence RKKRRQRRRPADRE on the Th cell epitope in Toxoid,tetanus source; Described aminoacid sequence 2 is 48-60 aminoacid sequence GRKKRRQRRRPPQ or 49-57 aminoacid sequence RKKRRQRRR or 48-57 aminoacid sequence GRKKRRQRRR in the HIV-1 TAT albumen; Described aminoacid sequence 3 is 18-27 aminoacid sequence FLPSDFFPSV or the mixed sequence RKKRRQRRRFLPSDFFPSV on the HBV cAg; Described aminoacid sequence 4 is 14-26 aminoacid sequence DPRVRGLYFPAGG or 14-30 aminoacid sequence DPRVRGLYFPAGGVSFG or the mixed sequence RKKRRQRRRDPRVRGLYFPAGGVSFG on the B cell epitope in HBV Pre S2 source.
Joining peptide between each aminoacid sequence of the present invention is AAA, GGG or SSS; Connecting modification joining peptide KSS on the N end of its N terminal amino acid sequence of described polypeptide immunogen; On the α of the lysine residue of the N of described modification joining peptide KSS end or the ε amino respectively or simultaneously covalently bound palmitoyl.
Polypeptide immunogen of the present invention can have the multiple polypeptides sequence, and wherein preferred peptide sequence is:
CH 3(CH 2) 14COKSSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGV SFG (polypeptide 1),
CH 3(CH 2) 14COK (CH 3(CH 2) 14CO) SSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (polypeptide 2),
CH 3(CH 2) 14COKSSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVR GLYFPAGGVSFG (polypeptide 3),
CH 3(CH 2) 14COK (CH 3(CH 2) 14CO) SSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLY FPAGGVSFG (polypeptide 4),
CH 3(CH 2) 14COKSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ (polypeptide 5),
CH 3(CH 2) 14COK (CH 3(CH 2) 14CO) SSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ (polypeptide 6),
CH 3(CH 2) 14COKSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQR RRPPQ (polypeptide 7),
CH 3(CH 2) 14COK (CH 3(CH 2) 14CO) SSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRP PQ (polypeptide 8),
CH 3(CH 2) 14COKSSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGV SFG (polypeptide 9),
CH 3(CH 2) 14COK (CH 3(CH 2) 14CO) SSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG (polypeptide 10),
CH 3(CH 2) 14COKSSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVR GLYFPAGGVSFG (polypeptide 11),
CH 3(CH 2) 14COK (CH 3(CH 2) 14CO) SSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLY FPAGGVSFG (polypeptide 12),
CH 3(CH 2) 14COKSSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG (polypeptide 13)
CH 3(CH 2) 14COK (CH 3(CH 2) 14CO) SSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG (polypeptide 14),
CH 3(CH 2) 14COKSSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV (polypeptide 15) or
CH 3(CH 2) 14COK (CH 3(CH 2) 14CO) SSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV (polypeptide 16).
A kind of preparation method of polypeptide immunogen may further comprise the steps:
A. the described polypeptide immunogen-resin of polypeptide solid phase synthesis, described polypeptide immunogen-resin is represented the polypeptide immunogen with resin-bonded;
B. polypeptide immunogen-resin is carried out cracking, obtain lysate;
C. lysate is adopted the volume-exclusion chromatography to carry out the initial gross separation purifying, obtain polypeptide immunogen by reverse chromatography purification then.
Polypeptide immunogen is used for the treatment of HBV chronic infection persistent state and relevant Secondary cases liver cirrhosis, the vaccine of liver cancer diseases or the application in the medicine in preparation.
Described HBV chronic infection persistent state is chronic hepatitis B or hepatitis b virus carrier.
Described vaccine or medicine also contain acceptable accessories, adjuvant and carrier.
The administering mode of described vaccine or medicine is a drug administration by injection.
The present invention's amino acid english abbreviation implication commonly used is:
A Ala L-Ala
D Asp aspartic acid
E Glu L-glutamic acid
F Phe phenylalanine
G Gly glycine
I Ile Isoleucine
K Lys Methionin
L Leu leucine
The acid of N Asn l-asparagine
P Pro proline(Pro)
Q Gln glutamy amino acid
R Arg arginine
S Ser Serine
T Thr Threonine
V Val Xie Ansuan
Y Tyr tyrosine
The present invention adopts the solid state chemistry synthetic method to prepare this kind immunogen, has determined optimum temps, feed ratio, peptide resin cleavage method and the factors such as time, purification condition of reaction system.
In the methods of the invention, when temperature of reaction during at 30-40 ℃, adopt HOBT/DCC coupling strategy, aminoacid sequence is synthetic continuously, and the optimum molar feed ratio of raw material and resin is 3: 1, and the average coupling efficiency of each amino acid or component reaches more than 99.5%; The trifluoroacetic acid method is adopted in the peptide resin cracking, and the best cracking time is 90 minutes.
Purifying adopts two-step approach: (1) adopts volume-exclusion chromatography preliminary purification, and dimethyl sulfoxide (DMSO) (DMSO) is a moving phase, and optimum column temperature is 20-40 ℃; (2) adopt the reversed phase chromatography method highly purified, studied its best factors such as batten spare, best chromatographic stuffing, optimum column temperature that go up.In the methods of the invention, best chromatographic stuffing is POROS 50 R1, and optimum column temperature is 22-34 ℃.
Through the comparison of ethanolic soln formulation, liposome turbid liquor body formulation, lipidosome freeze-dried formulation, prove that the liposome formulation inducing Th1 polarization and inducing CTL to be better than the ethanolic soln formulation aspect active, the subcutaneous injection pain obviously alleviates; Lipidosome freeze-dried formulation obviously is better than the liposome turbid liquor formulation aspect stable.
The present invention sets the lipidosome injection best prescription of described polypeptide immunogen.Wherein, contain aforementioned polypeptides immunogen, soybean phospholipid, palmitinic acid, vitamin-E and cholesterol.
Lipidosome freeze-dried formulation of the present invention also contains human albumin, N.F,USP MANNITOL and phosphoric acid salt except that containing aforementioned polypeptides immunogen, phosphatide, cholesterol, palmitinic acid, vitamin-E.
Polypeptide immunogen of the present invention and formulation thereof can be used any known immunization ways, for example: subcutaneous injection, intradermal injection, abdominal injection, intravenous injection etc.Immunizing dose can be 0.01nmol to 20nmol, can not use other adjuvant.Being used for immune HBV transgenic mice can the dose-dependently mode stimulate lymphocyte activation and propagation, and Th1 polarization and CTL reply, and suppress hbv replication, and HBV DNA copy number descends in the blood, and HbsAg and HbeAg titre descend or disappear.This renders a service to using single epitope polypeptide or multi-epitope polypeptide separately inaccessiable.
The present invention has determined to have the polypeptide immunogen of particular bone shelf structure based on Australia antigen(AA) epi-position collection of illustrative plates.This polypeptide immunogen can effectively excite Th1 polarization reaction, the reaction of HBV specific CTL and suppress duplicating of HBV in HBV transgenic mice body.Experimental result show the aforementioned polypeptides immunogen can be developed into into the treatment of a kind of novel hepatitis B and Secondary cases liver cirrhosis thereof, liver cancer with vaccine or medicine.
Embodiment
Following examples only are used to explain the present invention, but can not therefore limit the present invention.
Embodiment 1
The synthetic logical method of polypeptide immunogen
Adopt Fmoc solid state chemistry synthetic method.Fixedly carboxyl terminal extends peptide chain by the C end to the N end, carries out on synthesizer.Fmoc amino acid is raw material; applied sample amount is 0.75mmol; side chain protected amino acid Ser (tBU), Thr (tBU), Tyr (tBU), Gln (Trt), Asp (OtBU), Glu (OtBU), Arg (pbf), Asn (Trt), Lys (Boc); selecting the Wang-resin for use is solid phase carrier; applied sample amount 0.25mmol; raw material and resin mol ratio are 3: 1; first amino acid that is coupled on the resin adopts the coupling of DIC/HOBT method, and the HOBT/DCC method is adopted in the activation of all the other amino acid and palmitinic acid and coupling thereof.
Embodiment 2
Method is led in the polypeptide immunogen cracking
Select TFA lysate (0.75g phenol, 0.25ml dithioglycol, 0.5ml thioanisole, 0.5ml deionized water, 10.0mlTFA) for use, can suppress the generation of side reaction to greatest extent.Reaction density 40mg/ml (40mg refers to the quality of the embodiment 1 synthetic polypeptide immunogen-resin that obtains, and ml refers to the volume of lysate), temperature of reaction is 25 ℃, the reaction times is 1.5 hours.Peptide molecule is cut down from resin, and ether sedimentation, rotary evaporation get thick peptide product.
Embodiment 3
The polypeptide immunogen preliminary purification leads to method
Thick peptide product is adopted SEC application of sample 55.0ml, carry out the preliminary purification of peptide molecule.Chromatography condition: chromatographic system: P-6000 pump and AKTAeplorer 100; Chromatography column: diameter 25mm, column length 850mm; Filler Sephadex LH20; Moving phase: DMSO; Flow velocity: 2.0ml/min.Obtain purification result preferably.
Embodiment 4
The polypeptide immunogen purifying leads to method
Adopt chromatography column APS 50/275 POROS 50 R1 to carry out batch preparations.Behind peptide resin process cracking and the preliminary purification, the collection sample is divided into 7 times and carries out highly purified.Chromatography condition: chromatographic system: AKTAexplorer100; Sample concentration: 10.52mg/ml; Application of sample amount: 11.0ml; Chromatography column: diameter 50mm, column length 275mm; Filler POROS 50 R1; 34 ℃ of column temperatures; Mobile phase A: 30% ethanol-10mmol/L phosphoric acid; Mobile phase B: 90% ethanol-10mmol/L phosphoric acid; Gradient: 0-50%B, 5CV; 50-100%B, 0.5CV; 100-100%B, 0.5CV; Flow velocity: 100.0ml/min.Behind the elution peak amalgamation liquid mixing of collecting, deposit in-20 ℃ standby.The purity of this product is 98.5%, and yield is 85%.
Embodiment 5
Induce the Th1 polarization at the HBV transgenic mice
Adopt HBV-DNA transgenic mice (the full gene of ayw type HBV (1.3Kb) infects Kunming mouse).With the animal random packet, 10 every group, in the bilateral rib down and hind paw subcutaneous injection polypeptide 5, press 10,100 and 3 dosed administrations of 1000U/ mouse, weekly booster immunization once, totally three times.If IFN-α is 2b (15000U/ mouse) positive contrast medicine is established the negative contrast medicine of physiological saline.Finish back 10 days, 20 days, 30 days with last administration before the administration, get mouse spleen, separating spleen lymphocyte, with test product 10ng/ml stimulated in vitro 3 days, get supernatant, with the secretion situation of cytokines such as IFN-γ, IL-4 in the ELISA method detection culture supernatant, analyze and be subjected to inducing T cell Th1/Th2 type polar effect in the reagent object.The result can detect stronger IFN-γ secretion, and tangible dose-effect relationship is not seen in the detection of IL-4.
Embodiment 6
Induce the CTL activity at the HBV transgenic mice
Adopt HBV-DNA transgenic mice (the full gene of ayw type HBV (1.3Kb) infects Kunming mouse).With the animal random packet, 10 every group, in the bilateral rib down and hind paw subcutaneous injection polypeptide 5, press 10,100 and 3 dosed administrations of 1000U/ mouse, weekly booster immunization once, totally three times.If IFN-α is 2b (15000U/ mouse) positive contrast medicine is established the negative contrast medicine of physiological saline.Finish back 10 days, 20 days, 30 days with last administration before the administration, get mouse spleen, separating spleen lymphocyte is used test product 10ng/ml stimulated in vitro 3 days, detects the expression frequency of IFN-γ secretory cell in peripheral blood lymphocyte with the ELI-SPOT method.The result shows, finishing immunity back the 30th day, rising along with immunizing dose, the expression frequency of IFN-γ secretory cell raises in the peripheral blood lymphocyte, wherein 100 and the immunizing dose of 1000U/ mouse, induce the expression frequency of IFN-γ secretory cell in the peripheral blood lymphocyte to raise obviously the highest 3600 ± IFN-γ secretory cell/10 that detect in the body 6PBMC.
Embodiment 7
Suppress viral surface antigen (HbsAg) at the HBV transgenic mice
Adopt HBV-DNA transgenic mice (the full gene of ayw type HBV (1.3Kb) infects Kunming mouse).With the animal random packet, 10 every group, in the bilateral rib down and hind paw subcutaneous injection polypeptide 5, press 10,100 and 3 dosed administrations of 1000U/ mouse, weekly booster immunization once, totally three times.If IFN-α is 2b (15000U/ mouse) positive contrast medicine is established the negative contrast medicine of physiological saline.Finish back 10 days, 20 days, 30 days with last administration before the administration, get blood, separation of serum is measured HbsAg in the serum with the ELISA method respectively.The result shows that serum HbsAg obviously reduces, and is dose-dependently and time-dependent manner.
Embodiment 8
Suppress virus replication at the HBV transgenic mice
Adopt HBV-DNA transgenic mice (the full gene of ayw type HBV (1.3Kb) infects Kunming mouse).With the animal random packet, 10 every group, in the bilateral rib down and hind paw subcutaneous injection polypeptide 5, press 10,100,3 dosed administrations of 1000U/ mouse, weekly booster immunization once, totally three times.If IFN-α is 2b (15000U/ mouse) positive contrast medicine is established the negative contrast medicine of physiological saline.Finish back 10 days, 20 days, 30 days with last administration before the administration, get blood, separation of serum detects serum HBV DNA copy number with quantitative PCR method respectively.The result shows that serum HBV DNA copy number obviously reduces, and is dose-dependently and time-dependent manner.
Sequence table
＜110〉open loud, high-pitched sound
＜120〉a kind of polypeptide immunogen and its production and application
<130>
<160>16
<170>PatentIn?version?3.4
<210>1
<211>57
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉palmitoylation
<400>1
Lys?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Ser?Ser?Ser?Gly?Arg?Lys?Lys?Arg
1 5 10 15
Arg?Gln?Arg?Arg?Arg?Pro?Pro?Gln?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp
20 25 30
Phe?Phe?Pro?Ser?Val?Gly?Gly?Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr
35 40 45
Phe?Pro?Ala?Gly?Gly?Val?Ser?Phe?Gly
50 55
<210>2
<211>57
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉two palmitoylations
<400>2
Lys?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Ser?Ser?Ser?Gly?Arg?Lys?Lys?Arg
1 5 10 15
Arg?Gln?Arg?Arg?Arg?Pro?Pro?Gln?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp
20 25 30
Phe?Phe?Pro?Ser?Val?Gly?Gly?Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr
35 40 45
Phe?Pro?Ala?Gly?Gly?Val?Ser?Phe?Gly
50 55
<210>3
<211>66
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉brown paulownia acidylate
<400>3
Lys?Ser?Ser?Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr
1 5 10 15
Glu?Ala?Ala?Ala?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro?Pro
20 25 30
Gln?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Gly?Gly
35 40 45
Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Val?Ser
50 55 60
Phe?Gly
65
<210>4
<211>66
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉two palmitoylations
<400>4
Lys?Ser?Ser?Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr
1 5 10 15
Glu?Ala?Ala?Ala?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro?Pro
20 25 30
Gln?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Gly?Gly
35 40 45
Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Val?Ser
50 55 60
Phe?Gly
65
<210>5
<211>50
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉palmitoylation
<400>5
Lys?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp
1 5 10 15
Phe?Phe?Pro?Ser?Val?Gly?Gly?Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr
20 25 30
Phe?Pro?Ala?Gly?Gly?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro
35 40 45
Pro?Gln
50
<210>6
<211>50
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉two palmitoylations
<400>6
Lys?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp
1 5 10 15
Phe?Phe?Pro?Ser?Val?Gly?Gly?Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr
20 25 30
Phe?Pro?Ala?Gly?Gly?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro
35 40 45
Pro?Gln
50
<210>7
<211>59
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉palmitoylation
<400>7
Lys?Ser?Ser?Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr
1 5 10 15
Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Gly?Gly
20 25 30
Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Gly?Arg
35 40 45
Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro?Pro?Gln
50 55
<210>8
<211>59
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉two palmitoylations
<400>8
Lys?Ser?Ser?Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr
1 5 10 15
Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Gly?Gly
20 25 30
Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Gly?Arg
35 40 45
Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro?Pro?Gln
50 55
<210>9
<211>57
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉palmitoylation
<400>9
Lys?Ser?Ser?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro?Pro?Gln
1 5 10 15
Ser?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp
20 25 30
Phe?Phe?Pro?Ser?Val?Gly?Gly?Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr
35 40 45
Phe?Pro?Ala?Gly?Gly?Val?Ser?Phe?Gly
50 55
<210>10
<211>57
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉two palmitoylations
<400>10
Lys?Ser?Ser?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro?Pro?Gln
1 5 10 15
Ser?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp
20 25 30
Phe?Phe?Pro?Ser?Val?Gly?Gly?Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr
35 40 45
Phe?Pro?Ala?Gly?Gly?Val?Ser?Phe?Gly
50 55
<210>11
<211>66
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉palmitoylation
<400>11
Lys?Ser?Ser?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro?Pro?Gln
1 5 10 15
Ser?Ser?Ser?Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr
20 25 30
Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Gly?Gly
35 40 45
Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Val?Ser
50 55 60
Phe?Gly
65
<210>12
<211>66
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉two palmitoylations
<400>12
Lys?Ser?Ser?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg?Arg?Pro?Pro?Gln
1 5 10 15
Ser?Ser?Ser?Gln?Tyr?Ile?Lys?Ala?Asn?Ser?Lys?Phe?Ile?Gly?Ile?Thr
20 25 30
Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val?Gly?Gly
35 40 45
Gly?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Val?Ser
50 55 60
Phe?Gly
65
<210>13
<211>50
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉palmitoylation
<400>13
Lys?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp
1 5 10 15
Phe?Phe?Pro?Ser?Val?Gly?Gly?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg
20 25 30
Arg?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Val?Ser
35 40 45
Phe?Gly
50
<210>14
<211>50
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉two palmitoylations
<400>14
Lys?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Ala?Ala?Ala?Phe?Leu?Pro?Ser?Asp
1 5 10 15
Phe?Phe?Pro?Ser?Val?Gly?Gly?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg?Arg
20 25 30
Arg?Asp?Pro?Arg?Val?Arg?Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Val?Ser
35 40 45
Phe?Gly
50
<210>15
<211>44
<212>PRT
＜213〉artificial sequence
<220>
<221>MOD_RES
<222>(1)..(1)
＜223〉palmitoylation
<400>15
Lys?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Gly?Gly?Gly?Asp?Pro?Arg?Val?Arg
1 5 10 15
Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg
20 25 30
Arg?Arg?Phe?Leu?Pro?SerAsp?Phe?Phe?Pro?Ser?Val
35 40
<210>16
<211>44
<212>PRT
＜213〉artificial sequence
<220>
<22l>MOD_RES
<222>(1)..(1)
＜223〉two palmitoylations
<400>16
Lys?Ser?Ser?Pro?Ala?Asp?Arg?Glu?Gly?Gly?Gly?Asp?Pro?Arg?Val?Arg
1 5 10 15
Gly?Leu?Tyr?Phe?Pro?Ala?Gly?Gly?Gly?Arg?Lys?Lys?Arg?Arg?Gln?Arg
20 25 30
Arg?Arg?Phe?Leu?Pro?Ser?Asp?Phe?Phe?Pro?Ser?Val
35 40

Claims (10)

1. polypeptide immunogen, it is characterized in that, described polypeptide immunogen contains a peptide sequence, described peptide sequence contains aminoacid sequence 1, aminoacid sequence 2, aminoacid sequence 3 and aminoacid sequence 4, wherein, the joining peptide of being made up of several amino-acid residues between each aminoacid sequence connects or is directly covalently bound; Described aminoacid sequence 1 is the link to each other mixed sequence of formation of Th cell epitope sequence or Th cell epitope sequence and the proteic alkaline regional sequence covalency of TAT in virus of AIDS HIV-1 source; Described aminoacid sequence 2 is the proteic alkaline regional sequences of TAT in virus of AIDS HIV-1 source; Described aminoacid sequence 3 is link to each other with the proteic alkaline regional sequence covalency of TAT in virus of AIDS HIV-1 source mixed sequences of formation of the CTL epitope sequences in the CTL epitope sequences in hepatitis B virus source or hepatitis B virus source; Described aminoacid sequence 4 is link to each other with the proteic alkaline regional sequence covalency of TAT in virus of AIDS HIV-1 source mixed sequences of formation of the B cell epitope sequence in the B cell epitope sequence in hepatitis B virus source or hepatitis B virus source.
2. polypeptide immunogen according to claim 1 is characterized in that, described peptide sequence holds the ordering of C end to be from N:
Aminoacid sequence 1-aminoacid sequence 2-aminoacid sequence 3-aminoacid sequence 4,
Aminoacid sequence 1-aminoacid sequence 3-aminoacid sequence 4-aminoacid sequence 2,
Aminoacid sequence 1-aminoacid sequence 4-aminoacid sequence 2-aminoacid sequence 3,
Aminoacid sequence 2-aminoacid sequence 1-aminoacid sequence 3-aminoacid sequence 4,
With aminoacid sequence 1-aminoacid sequence 3-aminoacid sequence 4,
Wherein: when aminoacid sequence 2 existed, aminoacid sequence 1,3,4 can not be mixed sequence;
When ordering is aminoacid sequence 1-aminoacid sequence 3-aminoacid sequence 4, aminoacid sequence 1,
At least contain a mixed sequence in aminoacid sequence 3, the aminoacid sequence 4.
3. polypeptide immunogen according to claim 1 and 2, it is characterized in that described aminoacid sequence 1 is 830-843 aminoacid sequence QYIKANSKFIGITE or general Th cell epitope PADRE or mixed sequence RKKRRQRRRQYIKANSKFIGITE or the mixed sequence RKKRRQRRRPADRE on the Th cell epitope in Toxoid,tetanus source; Described aminoacid sequence 2 is 48-60 aminoacid sequence GRKKRRQRRRPPQ or 49-57 aminoacid sequence RKKRRQRRR or 48-57 aminoacid sequence GRKKRRQRRR in the HIV-1TAT albumen; Described aminoacid sequence 3 is 18-27 aminoacid sequence FLPSDFFPSV or the mixed sequence RKKRRQRRRFLPSDFFPSV on the HBV cAg; Described aminoacid sequence 4 is 14-26 aminoacid sequence DPRVRGLYFPAGG or 14-30 aminoacid sequence DPRVRGLYFPAGGVSFG or the mixed sequence RKKRRQRRRDPRVRGLYFPAGGVSFG on the B cell epitope in HBV Pre S2 source.
4. polypeptide immunogen according to claim 1 and 2 is characterized in that, the joining peptide between described each aminoacid sequence is AAA, GGG or SSS; Connecting modification joining peptide KSS on the N end of described polypeptide immunogen N terminal amino acid sequence; On the α of the lysine residue of the N of described modification joining peptide KSS end or the ε amino respectively or simultaneously covalently bound palmitoyl.
5. polypeptide immunogen according to claim 1 is characterized in that, described peptide sequence is:
CH 3(CH 2) 14COKSSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COK(CH 3(CH 2) 14CO)SSPADRESSSGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COKSSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COK(CH 3(CH 2) 14CO)SSQYIKANSKFIGITEAAAGRKKRRQRRRPPQAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COKSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ、
CH 3(CH 2) 14COK(CH 3(CH 2) 14CO)SSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ、
CH 3(CH 2) 14COKSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ、
CH 3(CH 2) 14COK(CH 3(CH 2) 14CO)SSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGGRKKRRQRRRPPQ、
CH 3(CH 2) 14COKSSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COK(CH 3(CH 2) 14CO)SSGRKKRRQRRRPPQSSSPADREAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COKSSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COK(CH 3(CH 2) 14CO)SSGRKKRRQRRRPPQSSSQYIKANSKFIGITEAAAFLPSDFFPSVGGGDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COKSSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COK(CH 3(CH 2) 14CO)SSPADREAAAFLPSDFFPSVGGGRKKRRQRRRDPRVRGLYFPAGGVSFG、
CH 3(CH 2) 14COKSSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV or
CH 3(CH 2) 14COK(CH 3(CH 2) 14CO)SSPADREGGGDPRVRGLYFPAGGGRKKRRQRRRFLPSDFFPSV。
6. preparation method as claim 1,2 or 5 described polypeptide immunogens is characterized in that may further comprise the steps:
A. the polypeptide solid state chemistry synthesizes polypeptide immunogen-resin,
B. adopt the trifluoroacetic acid method that polypeptide immunogen-resin is carried out cracking, obtain lysate,
C. lysate is adopted the volume-exclusion chromatography to carry out separation and purification, obtain polypeptide immunogen by reverse chromatography purification then.
7. be used for the treatment of the chronic persistent infection state of HBV and relevant Secondary cases liver cirrhosis, the vaccine of liver cancer diseases or the application in the medicine as claim 1,2 or 5 described polypeptide immunogens in preparation.
8. polypeptide immunogen as claimed in claim 7 is used for the treatment of the chronic persistent infection state of HBV and relevant Secondary cases liver cirrhosis, the vaccine of liver cancer diseases or the application in the medicine in preparation, it is characterized in that described HBV chronic infection persistent state is chronic hepatitis B or hepatitis b virus carrier.
9. polypeptide immunogen as claimed in claim 7 is used for the treatment of the chronic persistent infection state of HBV and relevant Secondary cases liver cirrhosis, the vaccine of liver cancer diseases or the application in the medicine in preparation, it is characterized in that described vaccine or medicine also contain acceptable accessories, adjuvant and carrier.
10. polypeptide immunogen as claimed in claim 7 is used for the treatment of the chronic persistent infection state of HBV and relevant Secondary cases liver cirrhosis, the vaccine of liver cancer diseases or the application in the medicine in preparation, and the administering mode that it is characterized in that described vaccine or medicine is a drug administration by injection.
CN200910227365A 2009-12-08 2009-12-08 Polypeptide immunogen, preparation method and application thereof Pending CN101717433A (en)

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WO2011069408A1 (en) * 2009-12-08 2011-06-16 Zhang Ga Polypeptide immunogen, preparation method and use thereof
CN106404731A (en) * 2016-08-30 2017-02-15 王燕 PCT (Procalcitonin) and CRP (C-Reactive Protein) double-label time resolution fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis
CN108283715A (en) * 2017-12-20 2018-07-17 江苏孟德尔基因科技有限公司 A kind of analogue antigen compound is for treating the related indication purposes of HBV infection
CN109575141A (en) * 2017-09-29 2019-04-05 苏州工业园区唯可达生物科技有限公司 A kind of CD4 helper T lymphocyte epitope fusogenic peptide and its vaccine

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Publication number Priority date Publication date Assignee Title
WO2011069408A1 (en) * 2009-12-08 2011-06-16 Zhang Ga Polypeptide immunogen, preparation method and use thereof
CN106404731A (en) * 2016-08-30 2017-02-15 王燕 PCT (Procalcitonin) and CRP (C-Reactive Protein) double-label time resolution fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis
CN106404731B (en) * 2016-08-30 2020-03-31 王燕 PCT and CRP double-labeling time-resolved fluorescence immunoassay method for simultaneously detecting bacterial meningitis and viral meningitis
CN109575141A (en) * 2017-09-29 2019-04-05 苏州工业园区唯可达生物科技有限公司 A kind of CD4 helper T lymphocyte epitope fusogenic peptide and its vaccine
CN108283715A (en) * 2017-12-20 2018-07-17 江苏孟德尔基因科技有限公司 A kind of analogue antigen compound is for treating the related indication purposes of HBV infection
WO2019120112A1 (en) * 2017-12-20 2019-06-27 江苏孟德尔基因科技有限公司 Use of mimetic antigen compound for treating symptoms related to hbv infections

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