CN109381698A - Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine - Google Patents

Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine Download PDF

Info

Publication number
CN109381698A
CN109381698A CN201710663647.6A CN201710663647A CN109381698A CN 109381698 A CN109381698 A CN 109381698A CN 201710663647 A CN201710663647 A CN 201710663647A CN 109381698 A CN109381698 A CN 109381698A
Authority
CN
China
Prior art keywords
ifn
alpha interferon
hepatitis
hypotype
human alpha
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201710663647.6A
Other languages
Chinese (zh)
Inventor
袁正宏
陈捷亮
李亚明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Fudan University
Original Assignee
Fudan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Fudan University filed Critical Fudan University
Priority to CN201710663647.6A priority Critical patent/CN109381698A/en
Priority to CN202210307193.XA priority patent/CN114917326A/en
Publication of CN109381698A publication Critical patent/CN109381698A/en
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5067—Liver cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/01—DNA viruses
    • G01N2333/02—Hepadnaviridae, e.g. hepatitis B virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Toxicology (AREA)
  • Cell Biology (AREA)
  • Virology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Wood Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Pathology (AREA)
  • Epidemiology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biophysics (AREA)
  • Communicable Diseases (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)

Abstract

The invention belongs to medicine and biotechnology fields, it is related to human alpha interferon hypotype and is preparing the application in anti-hepatic-B virus medicine, the present invention for the human genome coded interference element hypotype IFN-α 2 in the prior art for treating chronic hepatitis B response rate is lower and problem that side effect is larger, it provides through external virus transfection and infection model identification including IFN-α 14, a variety of alpha interferon hypotypes of IFN-α 6 and IFN-α 1, the test shows that, a variety of alpha interferon hypotypes compare IFN-α 2 with stronger anti-hepatitis B activity, and the acellular poison effect under antiviral concentration, wherein IFN-α 14 has strongest Anti-HBV effect.A variety of alpha interferon hypotypes of the present invention can be used for preparing the significant good anti-hepatic-B virus medicine of new anti-hepatitis virus effect.

Description

Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine
Technical field
The invention belongs to medicine and biotechnology fields, are related to the new pharmaceutical applications of Human alpha interferons, and in particular to people α Interferon is preparing the purposes in hepatitis B virus resisting medicine, the drug can for reducing or remove hepatitis B virus infection Viral surface antigen (HBsAg) and virus genom DNA (cccDNA) in liver cell.
Background technique
It is the important pathogen body for seriously endangering human health prior art discloses hepatitis type B virus (HBV).It is estimated that The target whole world about 2.4 hundred million HBV carrier, wherein nearly 80,000,000 people of the existing Patients with Chronic HBV Infection in China.Although existing at present The hepatitis B vaccine of HBV infection can effectively be prevented, but still have the newly-increased hepatitis B chronic infection case of hundreds of thousands every year, while is annual There is hundreds of thousands people to die of liver diseases caused by slow hepatitis B.Due to the shortage of specific treatment means, how to reach slow hepatitis B function Energy property is cured, i.e., hepatitis B surface antigen (HBsAg) is turned out cloudy, or even curing completely is virus genom DNA (cccDNA) removing, still It is the problem faced in the industry, in medical practice, many patients are control virus infection and cause a disease and need to take nucleosides (acid) class all the life Antiviral drugs, this causes serious financial burden to patient and its family and reduces the quality of life of patient.
Interferon (Interferon, IFN) was initially found and names in nineteen fifty-seven, was a kind of with direct disease-resistant toxic effect It answers, the cell factor of immunoregulation effect, plays key effect during host anti-virus immune response.It has identified at present IFNs have ten several, according to the difference of institute's bind receptor, IFNs is generally categorized into I and II amphitypy: I type interferon mainly includes IFN-α and IFN-β (IFN- λ is generally classified as type iii interferon), II type interferon are then mainly IFN-γ；Wherein, anti-with host Viral innate immunity it is closely related be IFN-α, by be specifically bound to cell surface IFNAR start downstream The transcriptional expression of the transduction of JAK-STAT signal path and then inducing interferon stimulated gene (ISGs) and play antivirus action. With the appearance and development of technique for gene engineering, recombinant human IFN-alpha is produced and is applied to the diseases such as treatment virus hepatitis.It grinds Study carefully display, compares another kind of for treating drug --- nucleosides (acid) analog, IFN-α and its PEGylated product of slow hepatitis B (PEG-IFN- α) also has both immunoregulation effect other than with direct antivirus action, therefore has higher hepatitis B surface Antigen (HBsAg) is turned out cloudy or even the function of serological conversion, and response continues more long in some patients；However, there is clinical research Statistics shows that the efficiency of IFN-α treatment slow hepatitis B still shows lower, even if being treated 48 months using long-acting interferon, HBe antigen (HBeAg) positive patient only can produce existing HBeAg and turn out cloudy for about three one-tenth, and the negative conversion rate of HBsAg is then lower than 5%, thus, it would be highly desirable to optimize Existing and exploitation novel hepatitis B means.
It is reported that existing 13 kinds of mankind's IFN-α hypotypes are identified that their encoding gene is equal successively including IFN-α 2 Positioned at No. 9 chromosomes of people.There is more similar structural domain, but having about 30% partial sequence is non-guarantor between each hypotype alpha interferon It keeps.The IFN-α 2 that more and clinical use is early stage identification discovery is studied at present, including IFN-α 2a/2b is (poor one between 2a and 2b A amino acid), and the function for remaining most IFN-α hypotype and its meaning in evolution and antiviral functions difference rarely have Research report.Although part report shows different subtype IFN-α by two subunit IFNAR1 with I type interferon receptors With IFNAR2 in conjunction with and play a role, but due to different with the affinity of two subunits, each hypotype IFN-α activation downstream is classical or other The mode and degree of road signal path have differences；Meanwhile different virus and different cells also deposit the sensibility of IFN hypotype In difference.
Status based on the prior art, the present invention for the comparison of anti HBV infecting effect between different subtype humanIFN-α through grinding Study carefully, it was found that the interferon hypotype of hepatitis B virus duplication can be significantly inhibited and mechanism is studied, intend providing mankind α interference The new pharmaceutical applications of element, and in particular to Human alpha interferons are preparing the purposes in hepatitis B virus resisting medicine, and the present invention is advantageous In promoting the understanding to alpha interferon Antiviral Mechanism in science, the slow second of novel therapeutic based on interferon hypotype is more developed Liver means lay the foundation.
Summary of the invention
Object of the present invention is to the statuses based on the prior art, provide the new pharmaceutical applications of Human alpha interferons, and the present invention passes through The comparative studies of Effect of anti-HBV between different subtype humanIFN-α, screening and identification 2 Effect of anti-HBV of IFN-α compared with the prior art Stronger humanIFN-α's hypotype, specifically, the present invention provides human alpha interferons to prepare the use in hepatitis B virus resisting medicine On the way, the present invention is conducive to promote the understanding to alpha interferon Antiviral Mechanism in science, and more exploitation is based on specific alpha interferon The new intervention slow hepatitis B means of hypotype lay the foundation.
The present invention is using HBV plasmid transfection liver cancer cell lines and HBV infection cell model to via 13 kinds of prokaryotic expression The antiviral effect of known mankind's IFN-α hypotype recombinant protein is screened, the results show that comparing used in clinic at present IFN-α 2, a variety of other IFN-α hypotypes have stronger Anti-HBV activity effect, and specific antiviral activity is ordered as 14 > α of IFN-α 1, α 6,α17,α21>α2,α5,α7,α8>α4,α10,α16。
Other IFN-α hypotypes of the present invention are other people genome encoding alpha interferon hypotypes in addition to IFN-α 2, Preferred IFN-α 14, α 1, α 6, α 17, α 21, α 5, α 7, α 8, α 4, α 10, α 16 in the present invention；Most preferably IFN-α 14 in the present invention.
The amino acid sequence of other IFN-α hypotype recombinant proteins of the present invention is obtained from human genome, amino acid Shown in sequence such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3.
The present invention using HBV infection primary hepatocyte system further comparison IFN-α 14, α 6 and IFN-α 2 Antiviral effect and difference, experiment confirm that IFN-α 14 and α 6 have the half of the inhibitory effect of HBeAg, HBsAg and HBV DNA Imitate inhibition concentration (IC50) low ten to hundred times of IFN-α 2 is compared, and it is dense in corresponding antiviral concentration and use identical with IFN-α 2 Equal no cytotoxicity effect under degree；Genomic DNA with the degradable HBV of IFN-α reported in the literature as transcription duplication template (cccDNA) compare, it is of the invention the results show that IFN-α 14, which compares IFN-α 2, has the stronger effect for lowering cccDNA.
Immunoblotting, interferon stimulate the reaction original part fluorescent reporter system and high flux gene chip of the present invention and Quantitative PCR data furthers elucidate, and compares IFN-α 2, specific IFN-α hypotype IFN-α 14 and 6 one side of α are to classical JAK- STAT1/STAT2 access has stronger activation effect, on the other hand has to bypass JAK=STAT3/STAT6 access relatively special Anisotropic activation effect；Meanwhile although induced in ISGs it is little with 2 difference of IFN-α on range, its inducing moiety reported with The relevant ISGs subgroup of efficacy of interferon therapy includes the effect in the isogenic generation of GBP5 with relative specificity.
The present invention the experimental results showed that, a variety of IFN-α hypotypes are generated inhibiting HBV surface antigen, e antigen and viral DNA In there is activity better than currently used IFN-α 2, wherein IFN-α 14 has strongest Anti-HBV effect, and equal acellular poison Effect, the present invention provides application foundation of the IFN-α hypotype in preparation treatment chronic hepatitis B newtype drug, especially preparing Reduce or remove the purposes in the drug of hepatitis B chronic infectious patients HBV surface antigen and cccDNA.
For ease of understanding, IFN-α 2 will be compared to a variety of humanIFN-α's hypotypes of the present invention by specific attached drawing below It is described in detail with more excellent Anti-HBV effect；It is noted that the attached drawing is merely to explanation, it is clear that this field it is general Logical technical staff can be according to illustrating herein, and make modification to the present invention within the scope of the invention, this hair is also included in these modifications In bright range.
Detailed description of the invention
The evaluation of each IFN-α hypotype Effect of anti-HBV and compare in Fig. 1 .HBV plasmid transfection system and HBV infection system,
Wherein, Figure 1A .HBV plasmid-transfected cells model；
HBV infection model of Figure 1B based on HepaRG cell；
Fig. 1 C. is based on the HBV infection model of human primary hepatocyte (PHH).
Fig. 2 .IFN- α 14, IFN-α 6 and IFN-α 2 inhibit the measurement of the half effective inhibition concentration of HBV and compare.
Fig. 3 .IFN- α 14 acts on the downward of HBV RNA and cccDNA.
Fig. 4 .IFN- alpha hypotype compares the activation effect of classical JAK-STAT1/STAT2 access and bypass.
Fig. 5 .IFN- α 14, the comparison in difference of IFN-α 6 and 2 inducement interferon stimulated gene of IFN-α spectrum.
Specific embodiment
1 HBV plasmid transfection system of embodiment
Each IFN-α hypotype recombinant protein processing prerotation is infected with to the cell of hepatitis B virus duplication plasmid, the table of hepatitis B The generation of face antigen (HBsAg), e antigen (HBeAg) and DNA (as shown in Figure 1A) is inhibited in various degree.
(1) culture of liver cancer cell lines Huh7: using DMEM culture solution, (Gibco company adds 10% fetal calf serum, 100U/ Ml penicillin, 100mg/ml streptomysin and 400mg/ml G418) in 5%CO2The lower 37 DEG C of constant temperature incubations of saturated steam environment；
(2) use the transfection reagent of Roche Holding Ag's production by hepatitis B virus duplication type plasmid pHBV1.3 (ayw serotype) etc. Import Huh7 cell；
(3) after cell culture 48 hours, it is separately added into each hypotype IFN-α (2ng/ml) to group of cells, is incubated for 72 hours； Wherein it should be noted that, because IFN-α 13 and the amino acid sequence of IFN-α 1 are completely the same, it are considered as isotype subclass, therefore detect in total 12 kinds of alpha interferon hypotypes；
(4) it collects cell conditioned medium ELISA and detects viral antigen marker HBeAg and HBsAg；Lytic cell, Southern Blot detects viral core particle DNA；
(5) there is the several alpha interferon hypotype such as IFN-α 14 and IFN-α 6 Anti-HBV activity better than IFN-α 2 to resist as the result is shown The effect that former and viral DNA generates；
(6) influence of each hypotype IFN-α to the activation of JAK-STAT access is further detected, the results show that 14 He of IFN-α The several alpha interferon hypotype such as IFN-α 6, which has, compares the stronger ability for activating classics JAK-STAT1/STAT2 of IFN-α 2 (such as Shown in Fig. 4 A).
2 HBV infection system of embodiment
By the processing of each IFN-α hypotype recombinant protein infected with the HepaRG (as shown in Figure 1B) or PHH of hepatitis B particle The generation of (as shown in Figure 1 C) cell, Hepatitis B virus e antigen (HBeAg) and DNA is inhibited in various degree.
(1) culture of HepaRG cell: HepaRG cell line derives from the terminally differentiated cells of people's liver precursor system, protects The many characteristics for having stayed primary human liver cell, can support HBV infection after differentiation；1) grown cultures liquid: 15% fetal calf serum, 100U/L penicillin, 0.1mg/L streptomysin, glutamine, 0.023IU/ml insulin, 4.7 μ g/ml hydrocortisones, 80 μ l/ Ml gentamicin；2) break up culture solution: grown cultures liquid+1.8%DMSO, need to continue differentiation two weeks or more just carry out HBV sense Dye；
(2) culture of PHH cell: purchased from the auspicious moral biology in Shanghai and dedicated commercial medium culture is used；It is noted that Human primary hepatocyte in research process of the present invention using three kinds of Different Individual sources has carried out testing and verifying correlated results, As a result with uniformity；
(3) it after cell infection 3 days, is separately added into each hypotype IFN-α (0.5 or 2ng/ml) to group of cells, it is every to be incubated for 72 Hour carries out changing liquid and reprocessing；
(4) it the 9th day after infecting, collects cell conditioned medium ELISA and detects viral antigen marker HBeAg, drawn with specific HBV Object qPCR detects the production of viral DNA in supernatant；
(5) the results show that there is the Anti-HBV activity better than IFN-α 2 to resist for IFN-α 14, IFN-α 6 and 1 alpha interferon hypotype of IFN-α The effect that former and viral DNA generates；It is in comprehensive rotaring redyeing system to be arranged as a result, obtaining the strong and weak of each alpha interferon hypotype Effect of anti-HBV 14 > α of sequence IFN-α 1, α 6, α 17,21 > α of α 2, α 5, α 7,8 > α of α 4, α 10, α 16；
(6) in the infection model based on PHH (as shown in Figure 2 A) and HepG2-NTCP (as shown in Figure 2 B), further with The IFN-α of various concentration handles cell, calculates half effective inhibition concentration (IC according to measured antiviral index50), as a result The IC of display, IFN-α 14 and IFN-α 6 to the inhibitory effect of HBeAg and HBV DNA50Low ten to hundred times of IFN-α 2 is compared, is shown With superior Anti-HBV effect；And CCK8 detects no cytotoxicity；
(7) it is further detected respectively with Northern Blot and qPCR and compares IFN-α 14 and IFN-α 2 to HBV RNA (such as Shown in Fig. 3 A) and cccDNA (as shown in Figure 3B) influence, the results show that IFN-α 14 compare IFN-α 2 have it is significantly stronger Inhibit HBV rna transcription and lowers the effect of cccDNA；
(8) Western Blot testing result further displays, and IFN-α 14 compares IFN-α 2 except can more effectively activate warp Outside allusion quotation JAK-STAT1/STAT2 access, also it can specifically activate JAK-STAT3/STAT6 by-passing signal logical relatively in low concentration Road；It plays a significant role in antiviral natural is immune in view of known STAT6 access, showing may be with the strong Anti-HBV activity of IFN-α 14 Effect is related；
(9) high flux gene chip have detected IFN-α 14, IFN-α 6 and IFN-α 2 handle HBV infection PHH cell 6 it is small When after full cellular gene expression (as shown in Figure 5), the results show that in IFN-α 14 and IFN-α 6 in interferon inducted gene (ISGs) induce is little with 2 difference of IFN-α on range, but induces at it and be higher than IFN-α 2 in amplitude, while its additional induction Lots of genes, clustered and functional analysis, the results show that IFN-α 14 can specifically be induced relatively and reported and interferon is treated Imitating relevant ISGs subgroup includes the genes such as GBP5, and wherein GBP5 gene is reported in antiviral process recently and plays important work With.
Experimental result prompt IFN-α 14 may by increase key ISGs molecule expression and simultaneously induce a group resist Virus activity molecule plays Effect of anti-HBV in turn, and the present invention illustrates its Anti-HBV activity for being much higher than IFN-α 2 from molecular mechanism level The possible cause of effect.
Screening and identification of the present invention effects of 13 kinds of alpha interferons in anti-hepatitis virus, the results showed that, a variety of alpha interferons Hypotype includes the Effect of anti-HBV for the IFN-α 2 that IFN-α 14, IFN-α 6 and IFN-α 1 have better than current clinical use, wherein 14 Effect of anti-HBV of IFN-α is most strong and can significantly lower viral surface antigen and cccDNA, and related mechanism may be with specific IFN- Alpha hypotype can have by the expression of the activation induced high levels key antiviral molecule of enhancing JAK-STAT classics and bypass path It closes；Of the present invention does not include that the human alpha interferon hypotype of IFN-α 2 can be used for preparing the hepatitis B virus resisting medicine washed.
SEQUENCE LISTING
<110>Fudan University
<120>human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine
<130> 20170806
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 166
<212> PRT
<213> IFN-α14
<400> 1
Cys Asn Leu Ser Gln Thr His Ser Leu Asn Asn Arg Arg Thr Leu Met
1 5 10 15
Leu Met Ala Gln Met Arg Arg Ile Ser Pro Phe Ser Cys Leu Lys Asp
20 25 30
Arg His Asp Phe Glu Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45
Gln Lys Ala Gln Ala Ile Ser Val Leu His Glu Met Met Gln Gln Thr
50 55 60
Phe Asn Leu Phe Ser Thr Lys Asn Ser Ser Ala Ala Trp Asp Glu Thr
65 70 75 80
Leu Leu Glu Lys Phe Tyr Ile Glu Leu Phe Gln Gln Met Asn Asp Leu
85 90 95
Glu Ala Cys Val Ile Gln Glu Val Gly Val Glu Glu Thr Pro Leu Met
100 105 110
Asn Glu Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Gln Arg Ile Thr
115 120 125
Leu Tyr Leu Met Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
130 135 140
Arg Ala Glu Ile Met Arg Ser Leu Ser Phe Ser Thr Asn Leu Gln Lys
145 150 155 160
Arg Leu Arg Arg Lys Asp
165
<210> 2
<211> 166
<212> PRT
<213> IFN-α6
<400> 2
Cys Asp Leu Pro Gln Thr His Ser Leu Gly His Arg Arg Thr Met Met
1 5 10 15
Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp
20 25 30
Arg His Asp Phe Arg Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45
Gln Lys Ala Glu Ala Ile Ser Val Leu His Glu Val Ile Gln Gln Thr
50 55 60
Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Val Ala Trp Asp Glu Arg
65 70 75 80
Leu Leu Asp Lys Leu Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
85 90 95
Glu Ala Cys Val Met Gln Glu Val Trp Val Gly Gly Thr Pro Leu Met
100 105 110
Asn Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr
115 120 125
Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
130 135 140
Arg Ala Glu Ile Met Arg Ser Phe Ser Ser Ser Arg Asn Leu Gln Glu
145 150 155 160
Arg Leu Arg Arg Lys Glu
165
<210> 3
<211> 166
<212> PRT
<213> IFN-α1
<400> 3
Cys Asp Leu Pro Glu Thr His Ser Leu Asp Asn Arg Arg Thr Leu Met
1 5 10 15
Leu Leu Ala Gln Met Ser Arg Ile Ser Pro Ser Ser Cys Leu Met Asp
20 25 30
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Asp Gly Asn Gln Phe
35 40 45
Gln Lys Ala Pro Ala Ile Ser Val Leu His Glu Leu Ile Gln Gln Ile
50 55 60
Phe Asn Leu Phe Thr Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Asp
65 70 75 80
Leu Leu Asp Lys Phe Cys Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
85 90 95
Glu Ala Cys Val Met Gln Glu Glu Arg Val Gly Glu Thr Pro Leu Met
100 105 110
Asn Ala Asp Ser Ile Leu Ala Val Lys Lys Tyr Phe Arg Arg Ile Thr
115 120 125
Leu Tyr Leu Thr Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
130 135 140
Arg Ala Glu Ile Met Arg Ser Leu Ser Leu Ser Thr Asn Leu Gln Glu
145 150 155 160
Arg Leu Arg Arg Lys Glu
165

Claims (7)

1. purposes of the human alpha interferon hypotype in preparation treatment chronic hbv-infection drug；The human alpha interferon hypotype It is selected from, human alpha interferon subtype alpha 14, α 1, α 6, α 17, α 21, α 5, α 7, α 8, α 4, α 10 and α 16.
2. purposes according to claim 1, which is characterized in that the human alpha interferon hypotype is human alpha interferon subtype alpha 14, encoding amino acid sequence is as shown in SEQ ID NO.1.
3. purposes according to claim 1, which is characterized in that the human alpha interferon hypotype is human alpha interferon hypotype IFN-α 6 and IFN-α 1, encoding amino acid sequence is respectively as shown in SEQ ID NO.2 and 3.
4. purposes according to claim 1, which is characterized in that the human alpha interferon hypotype recombinant protein, which has, to be inhibited Infect viral surface antigen (HBsAg), e antigen (HBeAg) virus protein and viral DNA and mRNA in the liver cell of hepatitis B The effect of generation.
5. purposes according to claim 1 or 2, which is characterized in that the human alpha interferon subtype alpha 14 is being used to prepare Reduce and remove the purposes in hepatitis B virus surface antigen (HBsAg) and genomic DNA drug.
6. purposes according to claim 5, which is characterized in that 14 recombinant protein of human alpha interferon subtype alpha reduce and Remove the viral surface antigen (HBsAg) in the liver cell of infection hepatitis B, e antigen (HBeAg) virus protein and viral DNA With the generation of mRNA, while lower virus transcription duplication the template i.e. content of genome covalently closed circular DNA (cccDNA), And the expression of relative specificity activation STAT6 and the interferon-stimulated gene GBP5 of induction tool Anti-HBV activity function.
7. purposes according to claim 1 or 2 or 3, which is characterized in that the human alpha interferon subtype alpha 14, α 6 and α 1 Anti-hepatitis B activity compared with 2 high ten times to hundred times of human alpha interferon subtype alpha.
CN201710663647.6A 2017-08-06 2017-08-06 Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine Pending CN109381698A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201710663647.6A CN109381698A (en) 2017-08-06 2017-08-06 Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine
CN202210307193.XA CN114917326A (en) 2017-08-06 2017-08-06 Application of human alpha interferon subtype in preparing anti-hepatitis B virus medicine

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710663647.6A CN109381698A (en) 2017-08-06 2017-08-06 Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine

Related Child Applications (1)

Application Number Title Priority Date Filing Date
CN202210307193.XA Division CN114917326A (en) 2017-08-06 2017-08-06 Application of human alpha interferon subtype in preparing anti-hepatitis B virus medicine

Publications (1)

Publication Number Publication Date
CN109381698A true CN109381698A (en) 2019-02-26

Family

ID=65412501

Family Applications (2)

Application Number Title Priority Date Filing Date
CN202210307193.XA Pending CN114917326A (en) 2017-08-06 2017-08-06 Application of human alpha interferon subtype in preparing anti-hepatitis B virus medicine
CN201710663647.6A Pending CN109381698A (en) 2017-08-06 2017-08-06 Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202210307193.XA Pending CN114917326A (en) 2017-08-06 2017-08-06 Application of human alpha interferon subtype in preparing anti-hepatitis B virus medicine

Country Status (1)

Country Link
CN (2) CN114917326A (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113209297A (en) * 2021-04-13 2021-08-06 中国人民解放军海军军医大学 Application of hepatitis B virus pregenomic RNA inhibitor in preparing medicine for treating hepatitis B related liver cancer
CN113817621A (en) * 2021-09-07 2021-12-21 天津大学 Recombinant saccharomyces cerevisiae strain capable of simultaneously expressing IFNa14 protein and human hepatitis B virus S protein as well as preparation method and application thereof

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115466802B (en) * 2022-10-28 2023-12-12 北京大学 Application of TP53BP2 in regulation and control of interferon signal path and antiviral

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1583795A (en) * 2003-06-30 2005-02-23 美国福源集团 Human interferon analogue with long-lasting biological effects
WO2006111745A2 (en) * 2005-04-20 2006-10-26 Viragen Incorporated Composition and method for treating viral infection
CN101244263A (en) * 2007-02-15 2008-08-20 赛德医药科技股份有限公司 Mixture containing mix interferon-alpha hypotype
CN101967196A (en) * 2010-11-10 2011-02-09 夏志南 Interferon fusion protein, preparation thereof and application thereof
WO2013059299A1 (en) * 2011-10-17 2013-04-25 The Uab Research Foundation Antibodies for interferon subtypes and interferon/interferon receptor ternary complex and uses thereof

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1583795A (en) * 2003-06-30 2005-02-23 美国福源集团 Human interferon analogue with long-lasting biological effects
WO2006111745A2 (en) * 2005-04-20 2006-10-26 Viragen Incorporated Composition and method for treating viral infection
CN101244263A (en) * 2007-02-15 2008-08-20 赛德医药科技股份有限公司 Mixture containing mix interferon-alpha hypotype
CN101967196A (en) * 2010-11-10 2011-02-09 夏志南 Interferon fusion protein, preparation thereof and application thereof
WO2013059299A1 (en) * 2011-10-17 2013-04-25 The Uab Research Foundation Antibodies for interferon subtypes and interferon/interferon receptor ternary complex and uses thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JIELIANG CHEN等: ""Functional Comparison of Interferon-α Subtypes Reveals Potent Hepatitis B Virus Suppression by a Concerted Action of Interferon-α and Interferon-γ Signaling"", 《HEPATOLOGY》 *
刘昕: ""干扰素治疗慢性乙肝不良反应观察和处理"", 《中国现代药物应用》 *
邬敏等: ""不同干扰素应答慢性乙型肝炎患者的基因组DNA甲基化谱差异分析"", 《微生物与感染》 *
陈彬: ""干扰素α-2b治疗乙型肝炎临床效果观察"", 《大家健康》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113209297A (en) * 2021-04-13 2021-08-06 中国人民解放军海军军医大学 Application of hepatitis B virus pregenomic RNA inhibitor in preparing medicine for treating hepatitis B related liver cancer
CN113817621A (en) * 2021-09-07 2021-12-21 天津大学 Recombinant saccharomyces cerevisiae strain capable of simultaneously expressing IFNa14 protein and human hepatitis B virus S protein as well as preparation method and application thereof
CN113817621B (en) * 2021-09-07 2023-02-17 天津大学 Recombinant saccharomyces cerevisiae strain capable of simultaneously expressing IFNa14 protein and human hepatitis B virus S protein as well as preparation method and application thereof

Also Published As

Publication number Publication date
CN114917326A (en) 2022-08-19

Similar Documents

Publication Publication Date Title
Yamamoto et al. Novel pH-sensitive multifunctional envelope-type nanodevice for siRNA-based treatments for chronic HBV infection
Mitra et al. Hepatitis B virus precore protein p22 inhibits alpha interferon signaling by blocking STAT nuclear translocation
Qiu et al. Long non-coding RNAs: regulators of viral infection and the interferon antiviral response
Ajiro et al. Oncogenes and RNA splicing of human tumor viruses
Tedeschi et al. Astrocytes produce interferon that enhances the expression of H-2 antigens on a subpopulation of brain cells.
Frese et al. Hepatitis C virus RNA replication is resistant to tumour necrosis factor-α
CN111499718B (en) Human alpha interferon receptor binding related site mutant and application thereof
Bausek et al. Cell interaction with a synthetic polynucleotide and interferon production in vitro
Schultz et al. Elimination of duck hepatitis B virus RNA-containing capsids in duck interferon-alpha-treated hepatocytes
CN109381698A (en) Human alpha interferon hypotype is preparing the purposes in hepatitis B virus resisting medicine
CN109021098B (en) Full Humanized monoclonal antibodies and its preparation method and application
CN101948845A (en) Optimization gene for coding chicken interferon alpha and application thereof in preparing chicken interferon alpha
Pellerin et al. Hepatitis C virus quasispecies variability modulates nonstructural protein 5A transcriptional activation, pointing to cellular compartmentalization of virus-host interactions
CN111592589B (en) Specific TCR for recognizing human hepatitis B virus core antigen C18-27 epitope
WO2019029081A1 (en) Application of interferon κ in the preparation of anti enveloped virus drugs
Fan et al. Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (WHV)
Miller et al. Evidence against a requisite role for defective virus in the establishment of persistent hepadnavirus infections.
Xu et al. Suppression of hepatitis B virus replication in Tupaia hepatocytes by tumor necrosis factor alpha of Tupaia belangeri
Nchioua et al. The Delta variant of SARS-CoV-2 maintains high sensitivity to interferons in human lung cells
Khabar et al. Hepatitis C virus-host interactions: the NS5A protein and the interferon/chemokine systems
CN107034201A (en) Apparent modification enzyme SETD2 antivirus action and its application
Moreau et al. Heterogeneity and functions of the 13 IFN‐α subtypes–lucky for some?
Lu et al. The interferon-α gene family of Marmota himalayana, a Chinese marmot species with susceptibility to woodchuck hepatitis virus infection
Xie et al. IFNα subtype-specific susceptibility of HBV in the course of chronic infection
Zhao et al. Cloning and characterization of porcine interferon-δ-related genes identified by genomic database screening

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination