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.

Active Publication Date: 2018-11-30
新兴县国研科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the indirect ELISA detection method for FAdV-4 antibody is prone to cross-reaction with other serotypes of avian adenovirus antibodies, and the specificity is poor
[0005] In view of the current situation that type 4 avian adenovirus has caused huge economic losses t

Method used

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  • Application of protein fiber2 and recombinant protein thereof in detection of 4-type fowl adenovirus antibody of serum
  • Application of protein fiber2 and recombinant protein thereof in detection of 4-type fowl adenovirus antibody of serum
  • Application of protein fiber2 and recombinant protein thereof in detection of 4-type fowl adenovirus antibody of serum

Examples

Experimental program
Comparison scheme
Effect test

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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Abstract

The invention discloses application of protein fiber2 and recombinant protein thereof in detection of a 4-type fowl adenovirus antibody of serum. According to the application, firstly, recombinant FAdV-4 fiber2 protein capable of being specifically combined with a 4-type fowl adenovirus specific antibody and a horse radish peroxidase labeled FAdV-4 enzyme-labeled antibody are obtained based on theprotein fiber2, and a method for specifically blocking the 4-type fowl adenovirus antibody in the serum and a detection kit are established by further using the recombinant protein as coating protein. The detection kit is high in sensitivity, high in specificity and good in stability, can be used for reflecting a neutralizing antibody level, is used for rapidly detecting the 4-type fowl adenovirus antibody in the serum, has the characteristics of simplicity in operation, high specificity, high sensitivity and low cost, can be used for simultaneously detecting a large number of samples and canbe used for monitoring an overall immunizing effect and neutralizing antibody level, so that powerful foundations and supports are provided for the diagnosis, vaccine prevention and control and the like of diseases, and the application has a very good application prospect.

Description

technical field [0001] The invention belongs to the technical field of breeding disease prevention and control. More specifically, it relates to the application of fiber2 protein and its recombinant protein in the detection of type 4 avian adenovirus (FAdV-4) antibody in serum by blocking ELISA method, a detection method and a kit. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAdV) is classified as Adenoviridae (Adenoviridae) fowl adenovirus (Aviadenovirus), according to the difference in antigenicity, divided into three groups, among them, group I adenovirus (FAdVI) has a common group Antigens, a total of 12 serotypes, widely exist in the eyes, upper respiratory tract and digestive tract of various poultry, most of which are recessive infection or cooperate with other diseases to cause disease or death of poultry. Since 2015, the incidence of avian adenovirus type 4 in chickens in my country has been on the rise, causing a disease characterized by perica...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/577G01N33/558
CPCG01N33/558G01N33/56983G01N33/577
Inventor 苏晓娜杨金易周庆丰蔡新斌曾道平李群辉罗洋洋李段陈丽
Owner 新兴县国研科技有限公司
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