CN113480643A - Preparation method of H5N8 subtype avian influenza virus HA polyclonal antibody - Google Patents

Preparation method of H5N8 subtype avian influenza virus HA polyclonal antibody Download PDF

Info

Publication number
CN113480643A
CN113480643A CN202110398897.8A CN202110398897A CN113480643A CN 113480643 A CN113480643 A CN 113480643A CN 202110398897 A CN202110398897 A CN 202110398897A CN 113480643 A CN113480643 A CN 113480643A
Authority
CN
China
Prior art keywords
influenza virus
avian influenza
polyclonal antibody
subtype avian
plasmid
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
CN202110398897.8A
Other languages
Chinese (zh)
Inventor
张晓璇
郭晶
常钦源
侯正阳
孙晓虹
梁晴
沈金艳
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.)
Liaocheng University
Original Assignee
Liaocheng 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 Liaocheng University filed Critical Liaocheng University
Priority to CN202110398897.8A priority Critical patent/CN113480643A/en
Publication of CN113480643A publication Critical patent/CN113480643A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • 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
    • C07K16/1018—Orthomyxoviridae, e.g. influenza virus
    • 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
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/16011—Orthomyxoviridae
    • C12N2760/16111—Influenzavirus A, i.e. influenza A virus
    • C12N2760/16151—Methods of production or purification of viral material
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Engineering & Computer Science (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Zoology (AREA)
  • Communicable Diseases (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Pulmonology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

The invention belongs to the fields of biotechnology and animal immunology, and relates to a preparation method of an HA polyclonal antibody of an H5N8 subtype avian influenza virus. The method comprises the following steps: deleting a cleavage site on an HA gene of the H5N8 subtype avian influenza virus, and then connecting the cleavage site to a eukaryotic expression vector to obtain a recombinant expression vector; after the recombinant expression vector is used for immunizing geese, the H5N8 subtype avian influenza virus HA polyclonal antibody is obtained through separation. Compared with the traditional whole inactivated vaccine, the H5N8 subtype avian influenza virus HA polyclonal antibody prepared by the invention not only ensures the safety of the test, but also induces the organism to generate cellular immunity and humoral immunity simultaneously, so that the organism can maintain the effective antibody level for a long time. The H5N8 subtype avian influenza virus HA polyclonal antibody HAs the advantages of simple preparation process, strong durability of immune effect, high safety and the like, and can provide long-acting immune protection for poultry infected by the H5N8 subtype avian influenza virus.

Description

Preparation method of H5N8 subtype avian influenza virus HA polyclonal antibody
Technical Field
The invention belongs to the fields of biotechnology and animal immunology, and relates to a preparation method of an HA polyclonal antibody of an H5N8 subtype avian influenza virus.
Technical Field
Influenza a viruses belong to the orthomyxoviridae family and are single-stranded negative-strand RNA viruses. Influenza viruses can be classified into 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) according to differences in Hemagglutinin (HA) and Neuraminidase (NA). Wherein, the two influenza viruses H17N10 and H18N11 are separated from bat bodies, the other influenza viruses of all subtypes can be separated from the bat bodies, and the wild waterfowls are the repositories of the viruses[1,2]. Avian Influenza Virus (AIV) belongs to influenza a virus, generally spherical or filamentous, and its genome includes surface genes HA and NA, Ribonucleoprotein (RNP) genes PB2, PB1, PA, nucleoprotein gene NP, matrix protein gene M, and nonstructural protein gene NS 8 RNA fragments. The HA protein can be combined with a sialic acid receptor on a host cell membrane, assists fusion of a virus envelope and the host cell membrane, plays an important role in a virus life cycle, HAs immunogenicity, and can form immune protection on an organism. H5 is firstly separated from Chinese wild duck in 2010Since the Highly Pathogenic Avian Influenza Virus (HPAIV) N8, it persists and has developed epidemic in the Asia-Pacific region, resulting in the death of large numbers of birds, causing significant losses to the poultry industry in many countries[3,4]. Therefore, monitoring H5N8 subtype AIV is not only beneficial to the healthy development of the poultry industry, but also has important significance to public health safety.
At present, methods for preventing and treating avian influenza mainly comprise monitoring, killing, harmless treatment, vaccine immunization and the like[5]. Whole virus inactivated vaccines are commonly used in vaccination, but this mode of vaccination can lead to difficulties in distinguishing immunized from naturally infected chickens, interfering with serological epidemiological monitoring. Therefore, the DNA vaccine has received much attention due to its high safety, simple preparation method, easy storage and transportation, and the like.
Disclosure of Invention
In order to solve at least one of the above problems in the prior art, the present study uses the currently popular H5N8 subtype highly pathogenic avian influenza virus in China as a representative strain, deletes the cleavage site of the HA sequence, connects to the eukaryotic expression vector pCAGGs, recombinates the expression plasmid immune geese, further prepares the polyclonal antibody, collects the serum antibody after the preparation is successful, and injects the serum antibody into the birds infected with the H5N8 subtype highly pathogenic avian influenza virus, thereby achieving the therapeutic purpose.
Specifically, the technical scheme of the invention is as follows:
the invention discloses a preparation method of an HA polyclonal antibody of H5N8 subtype avian influenza virus, which comprises the following steps:
deleting a cleavage site on an HA gene of the H5N8 avian influenza virus, and then connecting the cleavage site to a eukaryotic expression vector to obtain a recombinant expression vector; after the recombinant expression vector is used for immunizing geese, the H5N8 subtype avian influenza virus HA polyclonal antibody is obtained through separation.
Preferably, the HA gene and the plasmid are subjected to double enzyme digestion treatment by using restriction enzymes respectively, then the plasmid and the HA gene are connected by using ligase and are transformed into competent cells overnight for amplification, then enzyme digestion sequencing is carried out again, and the plasmid with the correct sequencing result is subjected to amplification culture.
In some embodiments of the invention, the HA gene and the plasmid pCAGGs are subjected to double digestion treatment by restriction enzymes EcoR I and Xho I, the plasmid and the HA gene are connected overnight by T4 ligase and then transformed into competent cells, after bacterial liquid identification, a positive bacterial colony is selected, amplified and cultured, and the plasmid is extracted, and the restriction enzymes EcoR I and Xho I are used for double digestion to obtain a target gene and sequencing; and (3) converting the positive plasmid with the correct sequencing result into competent cells for amplification culture, and preparing a large amount of recombinant expression vectors.
Preferably, the recombinant expression vector is injected into the leg of the goose, the intramuscular injection region is electrically stimulated, blood is collected from the heart after immunization, serum is collected, and the H5N8 subtype avian influenza virus HA polyclonal antibody is prepared.
In some embodiments of the invention, 180-; after immunization, the immunization is performed for 1 time at intervals of 25-35 days, and the immunization is performed for 3 times in total; and collecting blood from the heart 8-11 days after the three times of immunization, collecting serum, and preparing the HA polyclonal antibody of the H5N8 subtype avian influenza virus.
The invention also discloses a H5N8 subtype avian influenza virus HA polyclonal antibody prepared by the method.
The third aspect of the invention discloses an animal vaccine which comprises the HA polyclonal antibody of the H5N8 subtype avian influenza virus.
The fourth aspect of the invention discloses the application of the H5N8 subtype avian influenza virus HA polyclonal antibody in the preparation of a reagent for preventing avian influenza virus.
Preferably, the H5N8 subtype avian influenza virus HA polyclonal antibody is applied to preparation of a reagent for preventing H5N8 avian influenza virus.
The fifth aspect of the invention discloses the application of the method in preparing the reagent for preventing H5N8 avian influenza virus.
Compared with the prior art, the invention has the beneficial effects that:
the invention clones HA gene of H5N8 subtype AIV which is popular in China at present, and connects the HA gene to eukaryotic expression vector pCAGGs, and polyclonal antibody is obtained after goose immunization. The results of double enzyme digestion identification and sequencing show that the expression plasmid is successfully constructed. The success of the polyclonal antibody preparation is determined by an indirect fluorescence immunoassay and a Western blot test. And the specificity test result of the polyclonal antibody shows that the prepared H5N8 subtype avian influenza virus HA polyclonal antibody HAs strong specificity, good sensitivity and no cross reaction. Compared with the traditional whole inactivated vaccine, the H5N8 subtype avian influenza virus HA polyclonal antibody prepared by the invention not only ensures the safety of the test, but also induces the organism to generate cellular immunity and humoral immunity simultaneously, so that the organism can maintain the effective antibody level for a long time. The H5N8 subtype avian influenza virus HA polyclonal antibody successfully prepared by the invention HAs the advantages of simple preparation process, strong durability of immune effect, high safety and the like, and can provide long-acting immune protection for poultry damaged by H5N8 subtype AIV.
Drawings
FIG. 1 shows the restriction enzyme identification of expression plasmid pCAGGs-H5 (M: DNA molecular mass standard; 1: double restriction enzyme fragment of expression plasmid pCAGGs-H5; 2: expression plasmid pCAGGs-H5; 3: HA gene amplification fragment);
FIG. 2 shows the indirect immunofluorescence detection of polyclonal antibodies (A: expression plasmid pCAGGs-H5 transfected into 293T cells; B: empty vector pCAGGs transfected into 293T cells);
FIG. 3 shows the HA protein assay (1: transfection of expression plasmid pCAGGs-H5 into 293T cells; 2: transfection of empty vector pCAGGs into 293T cells; 3: 293T cells).
Detailed Description
The present application is further illustrated by the following detailed examples, which should be construed to be merely illustrative and not limitative of the remainder of the disclosure.
The instruments, equipment, reagents used in the examples are available from various sources, for example, purchased, or may be prepared.
Example 1
1.1 plasmids, cells and strains
According to the HA gene sequence of H5N8 subtype AIV in GenBank, the target gene is synthesized by Shanghai Czeri bioengineering GmbH. Competent cells were purchased from Beijing Quanji biology, Inc.; the eukaryotic expression vector pCAGGs is preserved by a livestock and poultry epidemic disease prevention and control technology innovation team of the college of agriculture of chat university; 293T cells were purchased from the cell bank of the Chinese academy of sciences.
1.2 Primary reagents
Ex Taq DNA polymerase, DL2000 DNA Marker and DL5000 DNA Marker were purchased from Takara Bio Inc. T4 DNA ligase, restriction enzymes EcoRI and XhoI were purchased from New England Biolabs. Gel recovery kits, plasmid extraction kits and purification kits were purchased from OMEGA. Liposome Lipofectamine 3000 was purchased from Invitrogen. DMEM and fetal bovine serum were purchased from GIBCO. Cell lysates were purchased from Thermo Scientific and SDS-PAGE protein loading buffer (2 ×) from shanghai bi yunnan biotechnology limited. FITC-labeled rabbit anti-chicken fluorescent antibody was purchased from Thermo Scientific, and horseradish peroxidase-labeled goat anti-chicken antibody was purchased from KPL.
1.3 construction and preparation of expression plasmid
The method comprises the steps of performing double enzyme digestion treatment on a target gene and a plasmid pCAGGs synthesized by a company by using restriction enzymes EcoR I and Xho I respectively, connecting the vector and the target gene overnight by using T4 ligase, transforming the connected vector and the target gene into competent cells, identifying bacterial liquid, selecting a positive bacterial colony, performing amplification culture, extracting a plasmid, performing double enzyme digestion by using the restriction enzymes EcoR I and Xho I again to obtain the target gene, and sequencing the target gene. And (3) converting the positive plasmid with the correct sequencing result into competent cells for amplification culture, and preparing a large amount of recombinant plasmids.
1.4 preparation of H5N8 subtype avian influenza Virus HA polyclonal antibody
6 gooses of 6 weeks old are taken, 200 mug of expression plasmid pCAGGs-H5 is injected into the muscle of each goose leg, and the intramuscular injection area is electrically stimulated to strengthen the stress response of the gooses. The booster immunization was performed 1 time 30 days after the immunization, and 3 times in total. And collecting serum after performing heart blood collection on the 10 th day after three times of immunization to obtain the H5N8 subtype avian influenza virus HA polyclonal antibody.
1.5 Indirect immunofluorescence assay
The expression plasmid pCAGGs-H5 is transfected to 293T cells, an indirect immunofluorescence test is carried out after transfection for 48H, and after an HA polyclonal antibody of H5N8 subtype avian influenza virus is used as a primary antibody for incubation, a rabbit anti-chicken secondary antibody with FITC markers is added.
1.6 Western blot detection
The expression plasmid pCAGGs-H5 is transfected to 293T cells, the cells are collected by centrifugation after 48 hours, an appropriate amount of cell lysate is added, the cells are incubated at 4 ℃ for 30min, an equal amount of 2 x protein loading buffer is added, and the cells are boiled in boiling water for 10 min. After SDS-PAGE runs glue and membrane transfer, a skim milk is used for sealing a membrane, after PBST is washed, the polyclonal antibody prepared in the step 1.5 is used as a primary antibody for incubation, after PBST is washed, a goat anti-chicken secondary antibody marked by horseradish peroxidase is used for incubation, and an Odyssey instrument is used for scanning to identify the effect of the polyclonal antibody.
1.7 evaluation of potency and specificity test of HA polyclonal antibody of H5N8 subtype avian influenza virus
The collected serum is subjected to hemagglutination inhibition test with H3, H4, H9 and H10 subtype AIV antigens and Newcastle Disease Virus (NDV) antigens, and the specificity and cross-reactivity of the HA polyclonal antibody of the H5N8 subtype avian influenza virus are tested.
2 results
2.1 construction of expression plasmid and restriction enzyme identification
After the HA gene of H5N8 is connected to the vector pCAGGs, the expression plasmid pCAGGs-H5 is digested for 2H at 37 ℃ by restriction enzymes EcoR I + Xho I to obtain an HA gene fragment (figure 1), the fragment is retrieved for sequencing, and the result shows that the construction of the expression plasmid is successful.
2.2 identification of H5N8 subtype avian influenza Virus HA polyclonal antibody
2.2.1 Indirect immunofluorescence assay
After the pCAGGs-H5 expression plasmid is transfected into 293T cells, specific green fluorescence is generated by detecting with a fluorescence microscope, and the 293T cells transfected with an empty vector can not detect the fluorescence (figure 2), which indicates that the eukaryotic expression plasmid can express HA protein of H5N8 AIV, and the target protein can be combined with antibodies in the prepared polyclonal antibody.
2.2.2 Western blot detection
After the expression plasmid pCAGGs-H5 is transfected by 293T cells, Western blot detection is carried out to find that HA protein is expressed at 75ku (figure 3), which shows that the HA protein can be combined with antibodies in the prepared polyclonal antibody, and the preparation of the polyclonal antibody is successful.
2.3 specificity test of HA protein polyclonal antibody of H5N8 subtype avian influenza virus
A laboratory-isolated A/Duck/Shandong/203/2017(H1N1), A/Chiken/Shandong/10/2018 (H3N2), A/Chiken/Shandong/423/2016 (H4N6), A/Chiken/Jiangxi/683/2016 (H5N1), A/Chiken/Shandong/1831/2016 (H6N2), A/Goose/Shandong/3/2019(H9N2), A/Chiken/Shandong/146/2018 (H10N6), A/Chiken/Shandong/23/2017 (H10N8) influenza viruses and Newcastle disease viruses NDV/Chiken/Shandong/2008/2017 were used to perform a hemagglutination inhibition test with the polyclonal H5N8 avian influenza virus HA antibody separately (Table 1), and only H5N1 was found to react with the polyclonal HA antibody, the antibody titer is 256, which indicates that the prepared H5N8 subtype avian influenza virus HA polyclonal antibody HAs no cross reaction with other subtypes of AIV.
TABLE 1 hemagglutination inhibition assay of polyclonal H5 antibody with other subtypes of AIVs and Newcastle disease Virus antigens
Figure BDA0003019532040000051
Discussion of 3
In recent years, the continuous prevalence and outbreak of avian influenza pose serious threats to the development of poultry industry and public health safety. In particular to highly pathogenic avian influenza of various subtypes, which not only infects poultry, but also possibly breaks through interspecific barrier transmission to people[6,7]. H5N8 HPAIV was first detected in chinese poultry in 2010 and first appeared in korean poultry in 1 month in 2014, and then the epidemic rapidly spread to many countries such as asia, europe and north america. It is a serious hazard to poultry farming and public health because it spreads rapidly across regions and can infect a wide variety of birds. In addition, Russia was reported to first develop human infections with H5N8 HPAIV at 2 months 2021. Therefore, work to enhance the study of the H5N8 avian influenza virus, in particular to enhance itThe prevention and treatment are particularly important.
The invention clones HA gene of H5N8 subtype avian influenza virus which is popular at home at present, and connects the HA gene to eukaryotic expression vector pCAGGs, and obtains polyclonal antibody after immunizing goose. The results of double enzyme digestion identification and sequencing show that the expression plasmid is successfully constructed. The success of the polyclonal antibody preparation is determined by an indirect fluorescence immunoassay and a Western blot test. And the specificity test result of the polyclonal antibody shows that the prepared H5N8 subtype avian influenza virus HA polyclonal antibody HAs strong specificity, good sensitivity and no cross reaction. Compared with the traditional whole inactivated vaccine, the H5N8 subtype avian influenza virus HA polyclonal antibody not only ensures the safety of the test, but also induces the organism to generate cellular immunity and humoral immunity simultaneously, so that the organism can maintain the effective antibody level for a long time. The H5N8 subtype avian influenza virus HA polyclonal antibody successfully prepared by the invention HAs the advantages of simple preparation process, strong durability of immune effect, high safety and the like, and provides long-acting immune protection for poultry damaged by H5N8 subtype AIV.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Reference to the literature
[1].Tong,S.,et al.,New world bats harbor diverse influenza Aviruses.PLoS Pathog,2013.9(10):p.e1003657.
[2].Wu,Y.,et al.,Bat-derived influenza-like viruses H17N10 and H18N11.Trends Microbiol,2014.22(4):p.183-91.
[3].Lee,Y.N.,et al.,Evaluation of the zoonotic potential of multiple subgroups of clade 2.3.4.4influenza A(H5N8)virus.Virology,2018.516:p.38-45.
[4].Kim,Y.I.,et al.,Genetic and phylogenetic characterizations of a novel genotype of highly pathogenic avian influenza(HPAI)H5N8 viruses in 2016/2017in South Korea.Infect Genet Evol,2017.53:p.56-67.
[5] Wan Xi white goose highly pathogenic avian influenza prevention, domestic animal veterinarian 2021.37(01): page 93.
[6] Piceida, study of the evolution and transmission laws of avian influenza virus, 2018, military academy of sciences page 114.
[7] Zhou Sen, the global spread and evolution of highly pathogenic avian influenza virus subtype H5 agricultural Notification in China 2019.35(02) page 104-.

Claims (10)

1. A preparation method of an HA polyclonal antibody of H5N8 subtype avian influenza virus is characterized by comprising the following steps:
deleting a cleavage site on an HA gene of the H5N8 avian influenza virus, and then connecting the cleavage site to a eukaryotic expression vector to obtain a recombinant expression vector; after the recombinant expression vector is used for immunizing geese, the H5N8 subtype avian influenza virus HA polyclonal antibody is obtained through separation.
2. The method of claim 1, wherein the HA gene and the plasmid are subjected to double enzyme digestion treatment by using restriction enzymes, the plasmid and the HA gene are connected by using ligase and then transformed into competent cells overnight for amplification, then enzyme digestion sequencing is performed again, and the plasmid with the correct sequencing result is subjected to amplification culture.
3. The method of claim 2, wherein the HA gene and the plasmid pCAGGs are subjected to double enzyme digestion treatment by restriction enzymes EcoR I and Xho I respectively, the plasmid and the HA gene are transformed into competent cells after being connected overnight by T4 ligase, after bacterial liquid identification, a positive colony is picked, the plasmid is enlarged and cultured and extracted, and the target gene is obtained and sequenced by double enzyme digestion by the restriction enzymes EcoR I and Xho I again; and (3) converting the positive plasmid with the correct sequencing result into competent cells for amplification culture, and preparing a large amount of recombinant expression vectors.
4. The method according to claim 1, wherein the recombinant expression vector is injected into the legs of a goose, the intramuscular injection region is electrically stimulated, blood is collected from the heart after immunization, serum is collected, and the H5N8 subtype avian influenza virus HA polyclonal antibody is prepared.
5. The method as claimed in claim 4, wherein 180 μ g of the recombinant expression vector is intramuscularly injected into each goose leg, and the intramuscular injection region is electrically stimulated to enhance the stress response of the goose; after immunization, the immunization is performed for 1 time at intervals of 25-35 days, and the immunization is performed for 3 times in total; and collecting blood from the heart 8-11 days after the three times of immunization, collecting serum, and preparing the HA polyclonal antibody of the H5N8 subtype avian influenza virus.
6. The H5N8 subtype avian influenza virus HA polyclonal antibody prepared according to the method of any one of claims 1-5.
7. An animal vaccine, characterized in that it comprises polyclonal antibody against HA of avian influenza virus subtype H5N8 according to claim 6.
8. The use of the H5N8 subtype avian influenza virus HA polyclonal antibody according to claim 6 in the preparation of an agent for preventing avian influenza virus.
9. The use of claim 8, wherein the H5N8 subtype avian influenza virus HA polyclonal antibody is used for preparing an agent for preventing H5N8 avian influenza virus.
10. Use of the method according to claims 1-5 for the preparation of an agent for the prevention of H5N8 avian influenza virus.
CN202110398897.8A 2021-04-14 2021-04-14 Preparation method of H5N8 subtype avian influenza virus HA polyclonal antibody Pending CN113480643A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110398897.8A CN113480643A (en) 2021-04-14 2021-04-14 Preparation method of H5N8 subtype avian influenza virus HA polyclonal antibody

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110398897.8A CN113480643A (en) 2021-04-14 2021-04-14 Preparation method of H5N8 subtype avian influenza virus HA polyclonal antibody

Publications (1)

Publication Number Publication Date
CN113480643A true CN113480643A (en) 2021-10-08

Family

ID=77932792

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110398897.8A Pending CN113480643A (en) 2021-04-14 2021-04-14 Preparation method of H5N8 subtype avian influenza virus HA polyclonal antibody

Country Status (1)

Country Link
CN (1) CN113480643A (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106039303A (en) * 2016-06-20 2016-10-26 华南农业大学 H5 subtype avian influenza DNA vaccine and preparation method thereof
RU2647566C1 (en) * 2017-06-06 2018-03-16 Федеральное государственное бюджетное учреждение "Федеральный центр охраны здоровья животных" (ФГБУ "ВНИИЗЖ") Strain a/goose/kalmykia/813/16 h5n8 virus avicum bird influenza virus avicum influenza type a and substitute h5 for control of antigen and immunizing activity of vaccine against avian influenza and for the manufacture of biopreparations for diagnostics and specific prevention of influenza birds of type a of substitute h5
CN109867722A (en) * 2018-08-16 2019-06-11 深圳出入境检验检疫局动植物检验检疫技术中心 AIV H5 hypotype monoclonal antibody, hybridoma and chromatograph test strip
CN110095593A (en) * 2019-04-28 2019-08-06 深圳贝安基因生物科技有限公司 A kind of double antibodies sandwich-enzyme-linked immunosorbent assay kit detecting H5N8 subtype avian influenza virus neuraminidase
KR20200085135A (en) * 2019-01-04 2020-07-14 대한민국(농림축산식품부 농림축산검역본부장) H5N8 strain Recombinant Influenza A virus and Vaccine Composition for H5 Serotype Influenza A virus belonging to clade 2.3.4.4A comprising the same

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106039303A (en) * 2016-06-20 2016-10-26 华南农业大学 H5 subtype avian influenza DNA vaccine and preparation method thereof
RU2647566C1 (en) * 2017-06-06 2018-03-16 Федеральное государственное бюджетное учреждение "Федеральный центр охраны здоровья животных" (ФГБУ "ВНИИЗЖ") Strain a/goose/kalmykia/813/16 h5n8 virus avicum bird influenza virus avicum influenza type a and substitute h5 for control of antigen and immunizing activity of vaccine against avian influenza and for the manufacture of biopreparations for diagnostics and specific prevention of influenza birds of type a of substitute h5
CN109867722A (en) * 2018-08-16 2019-06-11 深圳出入境检验检疫局动植物检验检疫技术中心 AIV H5 hypotype monoclonal antibody, hybridoma and chromatograph test strip
KR20200085135A (en) * 2019-01-04 2020-07-14 대한민국(농림축산식품부 농림축산검역본부장) H5N8 strain Recombinant Influenza A virus and Vaccine Composition for H5 Serotype Influenza A virus belonging to clade 2.3.4.4A comprising the same
CN110095593A (en) * 2019-04-28 2019-08-06 深圳贝安基因生物科技有限公司 A kind of double antibodies sandwich-enzyme-linked immunosorbent assay kit detecting H5N8 subtype avian influenza virus neuraminidase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
汪亮: "两株H5N8 亚型禽流感病毒生物学特性分析及其HA1蛋白抗原表位鉴定", 《中国优秀硕士学位论文全文数据库基础科学辑》, pages 141 *
黄璐 等: "H5亚型禽流感病毒DAS-ELISA检测方法的建立", 《泰山医学院学报》, no. 1, pages 14 - 18 *

Similar Documents

Publication Publication Date Title
Steel et al. Live attenuated influenza viruses containing NS1 truncations as vaccine candidates against H5N1 highly pathogenic avian influenza
Steel et al. Influenza virus vaccine based on the conserved hemagglutinin stalk domain
Krammer Emerging influenza viruses and the prospect of a universal influenza virus vaccine
Lee et al. Isolation and genetic characterization of H5N2 influenza viruses from pigs in Korea
Kim et al. Pathogenicity and vaccine efficacy of different clades of Asian H5N1 avian influenza A viruses in domestic ducks
CN101563361B (en) Influenza viruses able to infect canids, uses thereof
Avellaneda et al. Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus
Qiu et al. Protection against avian influenza H9N2 virus challenge by immunization with hemagglutinin-or neuraminidase-expressing DNA in BALB/c mice
Lee et al. Development and application of reference antisera against 15 hemagglutinin subtypes of influenza virus by DNA vaccination of chickens
Wu et al. Single dose of consensus hemagglutinin-based virus-like particles vaccine protects chickens against divergent H5 subtype influenza viruses
US11510976B2 (en) H7 avian influenza vaccine strain which differentiates infected from vaccinated animals, preparation method therefor, and application
Dundon et al. Progressive truncation of the Non-Structural 1 gene of H7N1 avian influenza viruses following extensive circulation in poultry
CN111849923A (en) Hybridoma cell 2D12 strain secreting monoclonal antibody against canine distemper virus H protein
US9504741B2 (en) Immune methods against influenza viruses and combinatorial vaccines thereof
Tung et al. Molecular characterization of a H5N1 highly pathogenic avian influenza virus clade 2.3. 2.1 b circulating in Vietnam in 2011
Shi et al. Single immunization with Newcastle disease virus-vectored H7N9 vaccine confers a complete protection against challenge with highly pathogenic avian influenza H7N9 virus
CN103169963B (en) Building and application of hydrophobia DNA (deoxyribonucleic acid) vaccine
Sączyńska et al. A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies
Sun et al. Development of an inactivated H7N9 subtype avian influenza serological DIVA vaccine using the chimeric HA epitope approach
CN114524862A (en) Construction and application of avian influenza (H5+ H7) trivalent DNA vaccine
CN112111503B (en) Adenovirus vector bivalent vaccine for simultaneously preventing H5 and H9 subtypes of avian influenza and preparation method thereof
CN103074306A (en) Newcastle disease virus rNDV-H52AH9 and construction method and application thereof
Peng et al. Protective efficacy of an inactivated chimeric H7/H5 avian influenza vaccine against highly pathogenic avian influenza H7N9 and clade 2.3. 4.4 H5 viruses
Liu et al. Development and evaluation of an avian influenza, neuraminidase subtype 1, indirect enzyme-linked immunosorbent assay for poultry using the differentiation of infected from vaccinated animals control strategy
CN113480643A (en) Preparation method of H5N8 subtype avian influenza virus HA polyclonal antibody

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