Application of protein fiber2 and recombinant protein thereof in detection of 4-type fowl adenovirus antibody of serum
A technology of fadv-4fiber2 and recombinant protein, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of poor specificity, economic loss of chicken industry, cross-reaction, etc.
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Embodiment 1
[0084] Example 1 Selection and preparation of FAdV-4 fiber2 protein
[0085]1. After analysis, hexon, penton and fiber are the main structural proteins, which constitute the nucleocapsid of avian adenovirus type 4. FAdV-4 has two fibers, one long fiber (fiber1) and one short fiber (fiber2). Among them, fiber2 is necessary for a certain stage of virus proliferation, assembly or diffusion, and the lack of fiber2 cannot produce virus particles. There are major species-specific epitopes and minor subgenus-specific epitopes on the fibers. Fibrin can recognize specific receptors on the cell membrane, that is, virus particles bind to cell receptors through fibrin to cause infection. In addition, fibrin is also antigenic. The recombinant fiber2 protein of the present invention is obtained through a baculovirus insect cell expression system.
[0086] 2. Protein preparation
[0087] (1) Sequence acquisition
[0088] The present invention carried out epidemiological monitoring on s...
Embodiment 2
[0101] Example 2 Animal challenge protection test
[0102] 1. Experimental materials
[0103] Experimental animals: 75 1-day-old SPF chickens without any vaccine for challenge.
[0104] Immunization vaccines: FAdV-4 whole virus inactivated vaccine, recombinant FAdV-4 fiber2 protein subunit oil vaccine, hexon protein subunit oil vaccine, PBS as control.
[0105] Challenge virus strain: FAdV-4 virus solution, PBS as control.
[0106] 2. Experimental method
[0107] Immunization age: 10 days old.
[0108] Age of challenge: 7 days after immunization.
[0109] The experimental animals were divided into inactivated vaccine, recombinant FAdV-4 fiber2, hexon, immunized blank control group and blank control group, with 15 animals in each group, immunized at 10 days old, and challenged at 1 week after immunization. The challenge control group used PBS as the immune control, and the blank group used PBS as the challenge and immune control. After the challenge, observe and record th...
Embodiment 3
[0115] Example 3 Preparation of enzyme-labeled antibody
[0116] (1) Screening of hybridoma cell lines
[0117] Five female BALB / c mice aged 6-8 weeks were selected, and the purified recombinant FAdV-4 fiber2 protein was diluted with PBS for three immunizations. One week after the third immunization, the blood was collected by docking the tail, and the FAdV-4 fiber2 protein was used as the coated antigen. The titer of the antiserum against the FAdV-4 fiber2 protein was determined by indirect ELISA method. When the titer was greater than 1:10 000, the mice reached fusion preparation The standard for monoclonal antibodies. Select 1-2 mice for cell fusion arrangement. Before fusion, the mice to be fused were euthanized by cervical dislocation, splenocytes were taken for fusion with SP2 / 0 myeloma cells, hybridoma cells were screened with HAT selective medium, and FAdV-4 fiber2 protein was used as the coating antigen. The positive hybridoma cells were screened by indirect ELISA ...
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