CN109134623A - A kind of epitope peptide of duck hepatitis A virus and its application - Google Patents

A kind of epitope peptide of duck hepatitis A virus and its application Download PDF

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CN109134623A
CN109134623A CN201811121417.8A CN201811121417A CN109134623A CN 109134623 A CN109134623 A CN 109134623A CN 201811121417 A CN201811121417 A CN 201811121417A CN 109134623 A CN109134623 A CN 109134623A
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virus
duck hepatitis
epitope peptide
duck
polypeptide
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岳华
汤承
王远微
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Southwest Minzu University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/02—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1009—Picornaviridae, e.g. hepatitis A virus
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/11—Immunoglobulins specific features characterized by their source of isolation or production isolated from eggs
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32422—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/32011—Picornaviridae
    • C12N2770/32411—Hepatovirus, i.e. hepatitis A virus
    • C12N2770/32434—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

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  • Gastroenterology & Hepatology (AREA)
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Abstract

The invention discloses a kind of epitope peptide of duck hepatitis A virus and its applications, and the amino acid sequence of epitope peptide is as shown in SEQ ID NO.4.The epitope peptide the preparation method is as follows: using the polypeptide of synthesis and KLH carrier protein is coupled by cysteine residues as mice immunized with antigen, Mouse spleen cells are taken to be merged with SP2/0 bone marrow cell, screening obtains hybridoma cell line, prepares monoclonal antibody；Neutralization test screening can neutralize the monoclonal antibody of gene C type and A type duck hepatitis A virus simultaneously in SPF duck embryos, measure the epitope peptide sequence that its corresponding hybridoma cell strain is identified, amino acid sequence is as shown in SEQ ID NO.4；Polypeptide shown in artificial synthesized SEQ ID NO.4 and KLH carrier protein are coupled as antigen Immune Laying Hens, extraction purification Yolk antibody.Yolk antibody of the present invention can be effectively prevented and treated disease caused by duck hepatitis A virus.

Description

A kind of epitope peptide of duck hepatitis A virus and its application
Technical field
The invention belongs to genetic engineerings and technical field of molecular biology, specifically, being related to a kind of duck hepatitis A virus Epitope peptide and its application.
Background technique
Duck virus hepatitis (Duck viral hepatitis, DVH) is by duck hepatitis virus (Duck hepatitis Virus, DHV) caused by within a kind of 3 week old of main harm duckling acute, height lethal infectious diseases.DHV includes tiny RNA Viraceae and Astroviridae member, and the DHV of Picornaviridae is named as duck hepatitis A virus (duck hepatitis A virus, DHAV), according to the difference of genotype structure, DHAV is divided into Gene A type (DHAV-A), Type B (DHAV-B) and c-type again (DHAV-C) three kinds of genotype.It is wherein Gene A type DHAV in the I type DHV of tradition of China's prevalence, Taiwan is new For gene Type B, and in recent years, South Korea's sample is novel or Area distribution of the DHAV of gene C type in China is relatively broad, gene A type and c-type DHAV prevalence while many duck culturing areas in China are that many ducks in China take for I traditional type DHV and arrange The basic reason of DVH still occurs after applying.
Therefore find the shared epitope of a Gene A type and c-type DHAV for duck virus hepatitis hyper-immune serum or The preparation of person's high immunity yolk antibody all has great importance for the prevention and control and emergency treatment of the disease.
Summary of the invention
In view of this, can be used for preparing the present invention provides a kind of epitope peptide of duck hepatitis A virus and its application The height that Gene A type and the infection of c-type duck hepatitis A virus can be neutralized simultaneously exempts from antibody.
In order to solve the above-mentioned technical problem, the invention discloses a kind of epitope peptide of duck hepatitis A virus, amino acid Sequence is as shown in SEQ ID NO.4 or the sequence is one or several amino acids formed with identical through replacement, missing or addition The amino acid sequence of immunogenicity and same antigen.
The invention also discloses a kind of epitope peptides of above-mentioned duck hepatitis A virus to prepare the application in inactivated vaccine.
The invention also discloses a kind of epitope peptides of above-mentioned duck hepatitis A virus to prepare the application in Yolk antibody.
The invention also discloses a kind of Yolk antibodies for preventing and treating duck hepatitis A virus disease, use during the preparation process Above-mentioned epitope peptide adds cysteine residues at the end C- of epitope peptide during synthetic antigen epitope peptide, uses The bis- property polypeptide coupling reagents of the SMPH of Thermo couple polypeptide fragment and KLH carrier protein by cysteine；Yolk antibody Potency is not less than 1:512.
The invention also discloses a kind of above-mentioned Yolk antibodies to prepare the medicine for preventing and treating duck hepatitis A virus disease Application in object.
Optionally, the duck hepatitis A virus is A type and c-type duck hepatitis A virus.
The invention also discloses a kind of preparation methods of the epitope peptide of above-mentioned duck hepatitis A virus, including following step It is rapid:
(1) synthesis polypeptide TKNIEDETVK, KWSRNHRPFR, NLFESKLTPY, TTHQIETVTI, PEIPKYDGPI, The end C- of every polypeptide of SSFKQDVMDQ, MDQIQSPSTV, PVLEIQPWGV, GVARMRAYTA adds cysteine residues；
(2) aforementioned polypeptides are mixed and is coupled by cysteine residues as antigen with KLH carrier protein；
(3) 6 week old BALB/c healthy mices are immunized in the antigen prepared, monoclonal antibody preparation process system routinely Standby and monoclonal antibody purification；
(4) with purifying obtain monoclonal antibody done in SPF duck embryos neutralization test screening simultaneously can in and gene C type with The monoclonal antibody of A type duck hepatitis A virus；
(5) the antigen polypeptide epitope that the monoclonal antibody with neutralization activity screened is identified i.e. SEQ ID NO.4 It is shown.
Compared with prior art, the present invention can be obtained including following technical effect:
Using the Yolk antibody of epitope peptide provided by the invention preparation, with safety, good, specific high, treatment is imitated The advantages such as fruit is good, production cost is low, can be effectively prevented and treated disease caused by duck hepatitis A virus, and application prospect is good.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technical effect.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, whereby to the present invention how application technology hand Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
Embodiment 1: the preparation of monoclonal antibody
The preparation of 1.1 antigens
TKNIEDETVK, KWSRNHRPFR, NLFESKLTPY, TTHQIETVTI, PEIPKYDGPI are synthesized, 9 polypeptides such as SSFKQDVMDQ, MDQIQSPSTV, PVLEIQPWGV, GVARMRAYTA, each 500 μ g of Peptide systhesis.
Cysteine residues are added at the end C- of polypeptide during synthesis polypeptide, with the bis- property polypeptide couplings of the SMPH of Thermo Joint-trial agent couples polypeptide fragment and KLH carrier protein by cysteine, as antigen.
Antigen coupling process is provided:
1,20mg SMPH is dissolved in 2ml DMF.
2,0.8ml KLH is added in 25ml round-bottomed flask, adding 1 × PBS (pH 7.2) makes final concentration of protein 15mg/ml。
3, the SMPH solution dissolved is slowly dropped in 120mg KLH albumen system, reaction 1h is stirred at room temperature.
4, it is dialysed 6 hours at 4 DEG C with 1L1 × PBS (PH 7.4) solution, removes free SMPH.
5, the KLH albumen after dialysis is poured into 50ml centrifuge tube, its volume is determined by the scale of centrifuge tube, according to anti- The amount for the KLH albumen being added before answering calculates the concentration of albumen after dialysis, then according to its concentration that 2.5mg KLH-SMPH is molten Liquid is transferred in 5ml centrifuge tube.
6, mixed polypeptide 1 × PBS of 0.6ml (pH 7.2) solution of 3.0mg synthesis is dissolved.
7, with the sulfydryl in Ellman reagent detection polypeptide: 100 μ l Ellman reagent stock liquid are added in 96 orifice plates, 10 μ l polypeptide solutions are added, survey its ultraviolet absorption value at λ=412nm with Nano spectrophotometer, if value > 0.15 OD is done In next step；OD value<0.15 is simultaneously>0.05 polypeptide is added, until reaching requirement；OD value < 0.05 returns to Peptide systhesis step matter again Control.
Ellman reagent is for detecting free sulfhydryl groups, if detection liquid displaing yellow illustrates that the sulfydryl of the Cys of polypeptide is big Part exists with free state；Illustrate that the sulfydryl in peptide C ys has been oxidized to form dimer if detection liquid not displaing yellow Or polymer.
8, polypeptide liquid is added drop-wise in KLH-SMPH pipe, mixes reaction 4 hours with vertical vortex mixer at room temperature.
9, with the sulfydryl in Ellman reagent detection polypeptide: 100 μ l Ellman reagent stock liquid are added in 96 orifice plates, Polypeptide solution after adding 10 μ l verification, measures ultraviolet absorption value at λ=412nm with Nano spectrophotometer.OD value < 0.03 illustrates that polypeptide and KLH protein-crosslinking rate have reached 80% or more；The activated KLH egg of SMPH is then added again in value > 0.03 OD It is white to continue to be crosslinked.If Ellman reagent displaing yellow illustrates that polypeptide and KLH albumen coupling are incomplete；If Ellman reagent is not shown Yellow then illustrate polypeptide all with KLH albumen coupling.
1.2 animal immune
6 week old BALB/c healthy mices are immunized in the antigen prepared.Antigen (100 microgram) and Freund's complete adjuvant 1:1 Mixing, intraperitoneal injection；Booster immunization 1 time after 2 weeks, antigen (100 microgram) are mixed with incomplete Freund's adjuvant 1:1, intraperitoneal injection； Primary every 1 week booster immunization later, the 4th enhances 100 micrograms antigen of inoculation, the 3rd day after being immunized, by mouse cervical dislocation It puts to death, it is sterile to take spleen for cell fusion.
The preparation and screening of 1.3 hybridoma cell strains
Cell-fusion techniques fusion routinely: it takes the spleen of immune mouse and SP2/0 bone marrow cell to be merged, is added The thymocyte and fused cell of mouse co-culture in HAT training system.
It using the polypeptide of synthesis as envelope antigen, is screened with indirect ELISA method and serial dilutions, by 3 times Cloning obtains the hybridoma of 10 plants of stably excreting MAb until all cloning cell hole Positive rates are 100% The epitope sequences of strain, this 10 strain of hybridoma strain identification are shown in Table 1.
The epitope sequences of 1 hybridoma of table identification
The preparation of 1.4 monoclonal antibody ascites
6~8 week old Balb/C mouse are taken, the sterile atoleine 0.5ml/ of intraperitoneal injection is only；After 1 week, intraperitoneal point It Zhu She not 10 plants of positive hybridoma cell strains；7~10 days after inoculation hybridoma cell strain, see that mouse web portion obviously expands, takes out abdomen Water, is collected after centrifugation supernatant, and -80 DEG C of crude products for freezing to obtain 10 monoclonal antibodies are spare.
Embodiment 2: in monoclonal antibody and the active identification of duck hepatitis A virus
Duck embryos median lethal dose (the LD of 2.1 gene C types and A type duck hepatitis A virus50) measurement
1) 12 age in days SPF duck embryos are divided into 11 groups, every group 5, wherein one group as a control group.
2) by gene C type and A type duck hepatitis A virus liquid respectively according to 10-1~10-10Do doubling dilution.
3) virus liquid of doubling dilution is inoculated into SPF duck embryos through allantoic cavity respectively, every injection 0.2ml, control group Inject the physiological saline of equivalent.
4) duck embryos of virus inoculation are put into 37 DEG C of incubators and are cultivated, every 6h observes duck embryos death condition, and notes down.
5) two kinds of viral duck embryos median lethal dose LD50 are calculated according to Reed&Muench method.
2.2 duck embryos neutralization tests
1) according to the measurement result of two kinds of viral duck embryos median lethal doses, with physiological saline by two kinds of viral dilutions at every .01ml contain 100LD50 in.
2) 10 monoclonal antibodies obtained in embodiment 1 are done into 2 times of doubling dilutions respectively, 1:2,1:4 ... .1: 2048。
3) by the virus diluted respectively with the monoclonal antibody mixed in equal amounts that has diluted, 37 DEG C of senses make 1h.
4) duck embryos are grouped, and the duck embryos of every kind of monoclonal antibody are respectively classified into 12 groups, every group 5,10 groups of experimental group, normal right According to 1 group and 1 group of virus control group of group.
5) mixture 5 pieces of duck embryos of inoculation, every injection 0.2ml are made in the sense of each dilution of experimental group.Normal group note Physiological saline 0.2ml is penetrated, virus control group injection 0.2ml contains 100 LD50Virus liquid.
6) duck embryos after inoculation are put into 37 DEG C of incubators and continue to cultivate 144h, discarded dead duck embryos, every 6h in for 24 hours and see Duck embryos death condition is examined, and is noted down, is shown in Table 2.
2 duck embryos death condition of table
7) each monoclonal antibody is calculated to two kinds of viral neutralization titers, experimental result according to Reed&Muench method It is shown in Table 3.
3 monoclonal antibody of table is directed to the neutralization titer of gene C type and A type duck hepatitis A virus
Antibody serial number Gene A type duck hepatitis A virus Gene C type duck hepatitis A virus
1 1:106 1:71
2 1:596 1:106
3 1:422 0
4 1:1189 1:841
5 0 1:89
6 1:355 0
7 0 1:106
8 1:355 1:178
9 1:71 1:708
10 1:355 1:89
8) No. 4 monoclonal antibodies can neutralize gene C type and A type duck hepatitis A virus simultaneously, and neutralization titer is respectively 1: 1189 and 1:841 is highest in all antibody, the best monoclonal antibody of neutralization, corresponding hybridoma cell strain (hybridoma cell strain is named as hybridoma cell strain 3L1-6, is stored in China typical culture collection center, preservation address For Wuhan University of Wuhan, China city, deposit number is CCTCC NO.C2013134, and the deposit date is on October 30th, 2013) know The amino acid sequence of other epitope is PVLEIQPWGV.
The preparation of 3 Yolk antibody of embodiment and clinical effectiveness test
The preparation of 3.1 antigens
PVLEIQPWGV polypeptide is synthesized, cysteine residues is added at the end C- of polypeptide during synthesis polypeptide, uses The bis- property polypeptide coupling reagents of the SMPH of Thermo couple polypeptide fragment and KLH carrier protein by cysteine, as antigen. The antigen prepared (1.6mg/ml) is mixed with Freund's complete adjuvant and incomplete Freund's adjuvant according to 1:1 respectively, uses threeway Syringe is emulsified, and prepares the inactivated vaccine containing Freund's complete adjuvant and incomplete Freund's adjuvant respectively.
The preparation of 3.2 Yolk antibodies
The bird inlay for taking 20 week old, using leg muscle and the subcutaneous multi-point injection of neck, head exempts to help completely using Freund Vaccinating agent 2ml/ only, is immunized after 14d, 28d, 42d with incomplete Freund's adjuvant vaccine after immune, and 2ml/, the 4th time It is immunized and starts in latter week to collect egg, measure duck hepatitis A virus antibody titer in yolk, collecting antibody titer is 1:256's or more Yolk liquid, by ammonium sulfate precipitation and dialysis purification Yolk antibody, Yolk antibody potency after purification is not less than 1:512.
The test of 3.3 clinical effectiveness
Duckling 80 that 1 age in days SPF duck embryos hatch are taken, breeding observing is randomly divided into 4 groups, every group 20,1 group after 2 days Inject duck hepatitis Yolk antibody prepared by the present invention, the duck hepatitis of 2 groups of injection Ruipu (Baoding) Biological Pharmaceutical Co., Ltd. production Yolk antibody (lot number: 201805113), the duck hepatitis Yolk antibody-of 3 groups of injection Shandong Sinder Technology Co., Ltd. production Duck is relaxed again, and (lot number: 201806211), 4 groups use physiological saline.1,2,3 groups of difference leg muscles inject Yolk antibody 0.8ml, and 4 Group same procedure injecting normal saline.The gene C type and A type duck hepatitis A virus of 10 LD50 are inoculated with after 24 hours respectively, often Then kind virus inoculation 10 observes the death condition of duckling in 7 days.
The different Yolk antibody clinical prophylactic performances of table 4 compare
Challenge viral dosage is carried out after immunization campaign in table 4, the yolk prepared using invention epitope peptide is anti- Gene A type and the c-type duck hepatitis A virus death rate after body are 0, hence it is evident that lower than other two groups 20%/30% and 20%/ 20%, and the death rate of control group is respectively 80% and 90%.It can be seen that the yolk prepared using epitope peptide of the present invention Antibody has good prophylactic treatment effect to Gene A type and c-type duck hepatitis A virus, and protective rate 100% is higher than existing in the market Commercialization Yolk antibody product.
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not It is confined to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification And environment, and can be carried out within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then it all should be in the appended power of invention In the protection scope that benefit requires.
Sequence table
<110>Southwest University for Nationalities
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Claims (7)

1. a kind of epitope peptide of duck hepatitis A virus, which is characterized in that its amino acid sequence as shown in SEQ ID NO.4, or The sequence is through replacement, missing or one or several amino acids formed ammonia with identical immunogenicity and same antigen of addition Base acid sequence.
2. the epitope peptide of duck hepatitis A virus described in claim 1 is preparing the application in inactivated vaccine.
3. the epitope peptide of duck hepatitis A virus described in claim 1 is preparing the application in Yolk antibody.
4. a kind of Yolk antibody for preventing and treating duck hepatitis A virus disease, which is characterized in that use right during the preparation process It is required that epitope peptide described in 1, residual plus cysteine at the end C- of epitope peptide during synthetic antigen epitope peptide Base is coupled polypeptide fragment and KLH carrier protein by cysteine with the bis- property polypeptide coupling reagents of the SMPH of Thermo；Yolk Antibody titer is not less than 1:512.
5. Yolk antibody described in claim 3 or 4 is preparing answering in the drug for preventing and treating duck hepatitis A virus disease With.
6. application according to claim 5, which is characterized in that the duck hepatitis A virus is A type and c-type duck hepatitis A virus.
7. the preparation method of the epitope peptide of duck hepatitis A virus described in claim 1, which is characterized in that including following step It is rapid:
(1) synthesis polypeptide TKNIEDETVK, KWSRNHRPFR, NLFESKLTPY, TTHQIETVTI, PEIPKYDGPI, The end C- of every polypeptide of SSFKQDVMDQ, MDQIQSPSTV, PVLEIQPWGV, GVARMRAYTA adds cysteine residues；
(2) aforementioned polypeptides are mixed and is coupled by cysteine residues as antigen with KLH carrier protein；
(3) by the antigen prepared be immunized 6 week old BALB/c healthy mices, routinely monoclonal antibody preparation process preparation and Monoclonal antibody purification；
(4) with purifying obtain monoclonal antibody done in SPF duck embryos neutralization test screening simultaneously can in gene C type and A type The monoclonal antibody of duck hepatitis A virus；
(5) the antigen polypeptide epitope that the monoclonal antibody with neutralization activity screened is identified is i.e. shown in SEQ ID NO.4.
CN201811121417.8A 2018-09-26 2018-09-26 A kind of epitope peptide of duck hepatitis A virus and its application Pending CN109134623A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111454337A (en) * 2020-03-04 2020-07-28 山东农业大学 Neutralizing mimic epitope shared by type 1 and type 3 duck hepatitis A virus and application thereof

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CN103059131A (en) * 2012-11-27 2013-04-24 天津市中升挑战生物工程有限公司 Method for preparing bivalent yolk antibody for preventing and treating duck virus hepatitis I and III
CN103820519A (en) * 2014-02-14 2014-05-28 西南民族大学 Monoclonal antibody of genetic C-type duck hepatitis A virus (DHAV) and applications thereof
CN104031145A (en) * 2014-04-16 2014-09-10 西南民族大学 Monoclonal antibody against duck hepatitis A virus type A and application thereof

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Publication number Priority date Publication date Assignee Title
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