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Establishment method of monoclonal antibody hybridoma cell strain for H3N2 canine influenza virus and preparation method and application of monoclonal antibody of monoclonal antibody hybridoma cell strain

A hybridoma cell line and monoclonal antibody technology, applied in the field of cell engineering, can solve problems such as unfavorable clinical research and development, and achieve the effects of high affinity, low risk and strong specificity

Active Publication Date: 2015-09-16
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Therefore, it is necessary to strengthen the research on CI; the Chinese patent application number 201410535918.6 discloses a monoclonal antibody against the H3N2 subtype canine influenza virus HA2 protein, which is obtained by preparing hybridoma cells from canine influenza virus isolate JS / 10, There are few studies on H3N2 canine influenza virus in the prior art, which is not conducive to clinical research and development

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  • Establishment method of monoclonal antibody hybridoma cell strain for H3N2 canine influenza virus and preparation method and application of monoclonal antibody of monoclonal antibody hybridoma cell strain
  • Establishment method of monoclonal antibody hybridoma cell strain for H3N2 canine influenza virus and preparation method and application of monoclonal antibody of monoclonal antibody hybridoma cell strain
  • Establishment method of monoclonal antibody hybridoma cell strain for H3N2 canine influenza virus and preparation method and application of monoclonal antibody of monoclonal antibody hybridoma cell strain

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Embodiment 1

[0033] The preparation of embodiment 1 hybridoma cell line

[0034] 1. Antigen preparation of H3N2 subtype canine influenza virus

[0035] The strain A / canine / Guangdong / 1 / 2006 (H3N2) (abbreviated as CGD1) used in the present invention has the Genbank accession numbers GU433345.1, GU433346.1, GU433347.1, GU433348.1, GU433349.1, GU433350 .1, GU433351.1, GU433352.1, strains and their sequence reference website: http: / / www.ncbi.nlm.nih.gov / nuccore / ? term=A%2Fcanine%2FGuangdong%2F1%2F 2006%28H3N2%29.

[0036] 1. Virus expansion culture: Inoculate the allantoic cavity of 10-day-old SPF chicken embryos with the diluted chicken embryo allantoic fluid of CGD1 strain, the inoculation volume is 0.15mL / embryo, and incubate at 37°C. Observe once every 12 hours, discard the dead chicken embryos within 24 hours, and store the dead embryos after 24 hours in a refrigerator at 4°C. After 96 hours, all the chicken embryos were cooled overnight at 4°C, the allantoic fluid of the chicken embryo...

Embodiment 2

[0092] The preparation of embodiment 2 monoclonal antibody

[0093] Female BALB / c mice were intraperitoneally injected with FICA, 0.5 mL / mouse. After 7 days, the hybridoma cells 2C5 in the logarithmic growth phase were suspended and washed with sterilized physiological saline (take the supernatant of the cell culture fluid in the logarithmic growth phase to measure the McAb titer), and the cell density was adjusted to 2×10 6 individual / mL. Each mouse was intraperitoneally injected with 0.5mL cell suspension, and observed whether the abdomen of the mouse was swollen day by day. When the ascites was obviously swollen, the ascites was taken out, placed in an EDTA-treated anticoagulant tube, and centrifuged at 5,000r / min for 10min to remove the cells. precipitation. The collected supernatant was inactivated at 56° C. for 30 minutes, centrifuged at 10,000 r / min for 15 minutes, and then purified. Do a good job of disinfection before and after the extraction of mouse ascites, and ...

Embodiment 3

[0128] Example 3 Establishment of Double Antibody Sandwich ELISA Detection Method

[0129] Preparation of rabbit-derived polyclonal antibody: Take two 3kg New Zealand white rabbits, phacoemulsify with purified H3N2CIV and the same amount of FCA, and inject subcutaneously in multiple points on the back, 1.5mg / rat. The second and third immunizations were emulsified with FICA and H3N2CIV on the 14th day and 28th day after the first immunization, respectively, and injected subcutaneously as the antigen, 2 mg per mouse. After the third immunization, blood was collected from the heart, the serum was separated, and stored at -20°C for future use. The titer of the antiserum was determined by indirect ELISA, and the polyclonal antibody concentration after purification (purification steps were the same as in Example 2) was measured using a micro-spectrophotometer, and the purified samples were stained with Coomassie brilliant blue to detect the purification effect.

[0130] Table 12 Pr...

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Abstract

The invention relates to the technical field of cell engineering and discloses an establishment method of a monoclonal antibody hybridoma cell strain for an H3N2 canine influenza virus and a preparation method and application of a monoclonal antibody of the monoclonal antibody hybridoma cell strain. The monoclonal antibody hybridoma cell strain is preserved in China Center for Type Culture Collection on May 13, 2015; the preservation number is CCTCC C201555. The monoclonal antibody secreted by the monoclonal antibody hybridoma cell strain is combined with specificity of hemagglutinin of the H3N2 canine influenza virus and is high in appetency; the titer after purification is 1: 320,000, and the monoclonal antibody does not react with CDV, CPV, CPIV, CAV-II, CRV, H7N9, H10N8 or H9N2 subtype influenza viruses. By injecting the monoclonal antibody used for preventing and controlling the infection of the influenza viruses, compared with a traditional vaccination method, the method is higher in specificity, lower in risk and higher in safety, and has the actual practical value.

Description

technical field [0001] The invention relates to the technical field of cell engineering, and more specifically relates to the establishment of a monoclonal antibody hybridoma cell line of H3N2 canine influenza virus and the preparation and application of the monoclonal antibody. Background technique [0002] Canine influenza (Canine influenza, CI) is a canine animal contact respiratory infectious disease caused by canine influenza virus (CIV), type A influenza virus (IAV) of Orthomyxoviridae family. The disease first broke out in the United States in 2004, and was subsequently reported in Eurasia and other regions. CI is mainly divided into two subtypes, H3N2 and H3N8. The H3N8 subtype is mainly used in Europe and the United States, and the H3N2 subtype is mainly used in most Asian countries. Over the past ten years, IAV has continuously broken through interspecies constraints and cross-infected humans and animals, which has aroused widespread concern about influenza. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/577G01N33/569A61K39/42A61P31/16C12R1/91
Inventor 李守军李陆涛袁子国孙凌霜贾坤远立国卢刚涂黎晴
Owner SOUTH CHINA AGRI UNIV
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