H5 subtype avian flu virus hemagglutinin protein monoclonal antibody, and its preparing method and use
A technology of monoclonal antibody and avian influenza virus, applied in antiviral immunoglobulin, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems that cannot meet the needs of clinical testing
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Embodiment 1
[0042] Embodiment 1: Preparation of anti-H5 subtype avian influenza virus HA gene monoclonal antibody
[0043] 1) Antigen preparation:
[0044] Nine-day-old fertilized chicken embryos were inoculated with the H5N1-Yu22 strain virus, incubated at 30°C for 2 days, and the chicken embryo liquid was collected to obtain the amplified H5N1-Yu22 strain virus. Collect the live virus and inactivate it with 0.03% formalin at 4°C. The inactivated virus is detected by HA to determine the titer of the inactivated virus solution (Note: For the specific methods of HA titer determination and HI detection, refer to WHO operation guideline, we chose HA=1024, which was provided by the Department of Microbiology, University of Hong Kong).
[0045] 2) White mice: female, 6-week-old Balb / c mice were purchased from the Anticancer Center of Xiamen University, and were bred in the center for experiments.
[0046] 3) Preparation of hybridoma:
[0047] We use standard in vivo immunization methods and...
Embodiment 2
[0060] Example 2: Preparation of H5 Subtype Influenza Virus HA Recombinant Antigen
[0061] 1) Bacmid-H5HA recombinant virus obtained
[0062] Select the HA full-length cDNA gene of the avian influenza AIV-H5N1-Yu22 virus strain as a template (provided by the Department of Microbiology, University of Hong Kong), design a pair of primers (synthesized by Shanghai Boya), set BamHI at the 5' end, and EcoRI at the 3' end, and carry out PCR amplification, using gel recovery kit (Hua Shun company) to recover the PCR product, to obtain a DNA fragment of about 1.7kb, and then clone it into the pMD18-T vector (Dalian Bao Biological Company), and obtain the correct DNA fragment by enzyme digestion and sequencing. Cloning of plasmid pMD18-T-H5HA. Then the target fragment was digested with BamHI and EcoRI from pMD18-T-H5HA to obtain the H5HA gene, and then ligated into the pFastBacl vector, and the ligated material was transformed into CaCl 2 The sensitized E.coli.DH5α was identified to ...
Embodiment 3
[0068] Example 3: Assembly of H5 subtype influenza virus HA antigen detection kit (enzyme-linked immunoassay, ELISA).
[0069] 1) Principle:
[0070] This kit uses the double-antibody sandwich method to detect the HA antigen of the H5 subtype influenza virus in the specimen.
[0071] Firstly, the monoclonal antibody against H5 influenza virus HA gene is pre-coated on the polyethylene microwell plate strip of the kit. When the cleaved H5 influenza virus HA antigen is added to the microwell, the pre-coated monoclonal antibody It can be captured, and then the enzyme-labeled monoclonal antibody added can also bind to it, and then the result can be judged by the degree of color development of the enzyme-catalyzed substrate. When the specimen does not contain influenza virus antigen or H5 influenza virus, the substrate will not develop color. Detectable specimens include excreta, oral and nasal secretions, whole virus or lysed virus cultured in chicken embryos, etc.
[0072] 2) P...
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