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Riemeria anatipestifer ompa/motb truncated recombinant protein, its antibody, preparation method and application

A technology of Riemeria anatipestifer and recombinant protein, applied in the field of genetic engineering, to achieve the effect of simple preparation method and realization of standardization

Active Publication Date: 2019-01-04
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since the research on Thrombospondin type 3 repeats: OmpA / MotB gene and its protein of Riemerella anatipestifer has not been reported at home and abroad, the research on Thrombospondin type 3 repeats: OmpA / MotB gene and its protein Research helps open up new ideas for RA prevention and control

Method used

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  • Riemeria anatipestifer ompa/motb truncated recombinant protein, its antibody, preparation method and application
  • Riemeria anatipestifer ompa/motb truncated recombinant protein, its antibody, preparation method and application
  • Riemeria anatipestifer ompa/motb truncated recombinant protein, its antibody, preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of truncated OmpA / MotB recombinant protein

[0032] 1. Primer design for OmpA / MotB gene (main epitope region)

[0033] According to the RA-CH-1 bioinformatics analysis and deduced OmpA / MotB gene sequence (Genbank ID number: CP003787.1) information published by Gene Bank, the OmpA / MotB gene fragment is 1467bp in length, and the protein epitope is analyzed using BepiPred 1.0bServer Analysis, further using Protean in DNAstar7.0 to analyze the sequence, select the main epitope segment of RA-CH-1 OmpA / MotB: the 409bp-1389bp segment (969bp) for truncated expression. Using the extracted RA-CH-1 genomic DNA as a template, the DNA fragment of the main antigen epitope region of OmpA / MotB gene was obtained by PCR amplification, the expected length (primer region plus restriction site) was 981bp, and the main antigen of OmpA / MotB The nucleotide sequence of the epitope region is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[003...

Embodiment 2

[0059] Example 2 Establishment of indirect ELISA method based on OmpA / MotB truncated protein

[0060] 1. Optimization of reaction conditions based on OmpA / MotB truncated protein ELISA method

[0061] The optimization conditions are shown in Table 1.

[0062] Table 1 Optimization of indirect ELISA conditions

[0063]

[0064]

[0065] Optimize according to the following steps with each reaction condition of table 1:

[0066] (1) Dilute the protein with coating solution, add 100 μL to each well of the microplate, and overnight at 4°C;

[0067] (2) Discard the liquid the next day, add 200 μL of PBST containing 1% BSA to each well, and block at 37°C for 1 hour;

[0068] (3) Wash with PBST 4 times, each time for 5 minutes, pat dry, add 100 μL of serum to be tested, and incubate at 37°C for 1 hour;

[0069] (4) Wash 4 times with PBST, pat dry, add 100 μL of HRP-labeled goat anti-duck IgG at working concentration, and incubate at 37°C for 1 hour;

[0070] (5) Wash the plat...

Embodiment 3

[0117] Example 3 Preparation of rabbit anti-OmpA / MotB truncated recombinant protein hyperimmune serum

[0118] 1. Preparation of immunogen

[0119] The OmpA / MotB truncated recombinant protein prepared in Example 1 was mixed with Freund's complete adjuvant (FCA) or Freund's incomplete adjuvant (FICA) in equal volumes, placed on an ice bath, and ultrasonically emulsified to form a water-in-oil agent until. Check the emulsification effect standard, that is, drop the emulsified antigen in ice water to form a complete and continuous non-diffusion round oil droplet and keep it for 1 minute.

[0120] 2. Animal immunity

[0121] Rabbits were immunized according to the immunization procedure in Table 16, blood was drawn from the rabbits one day before the first immunization, and serum was separated as negative serum control.

[0122] Table 16 Preparation of hyperimmune serum for rabbit anti-OmpA / MotB truncated recombinant protein immunization program

[0123]

[0124] 3. Prepara...

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Abstract

The invention discloses a Riemerella anatipestifer OmpA / MotB truncated recombinant protein, its antibody and a preparation method and an application thereof. The recombinant protein has immunoreactivity similar to natural OmpA / MotB protein, can bind to RA antibody specifically, will not generate cross reactions with Salmonella anatum positive serum, duck colibacilosis positive serum, duck swollen head septicemia virus positive serum, avian influenza virus (H5) positive serum, duck plague virus positive serum, duck hepatitis virus positive serum and the like, and can be used as a detection antigen for detecting whole bacteria antibody. As the recombinant protein is not whole bacteria, there is no risk of spreading bacteria when the recombinant protein is applied to detection. In addition, the preparation method of the recombinant protein is simple, is suitable for large-scale industrial production, can realize standardization of products and is beneficial to result comparison between different laboratories.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a Riemeria anatipestifer OmpA / MotB truncated recombinant protein, an antibody thereof, a preparation method and application. Background technique [0002] Riemerella anatipestifer (Riemerella anatipestifer, RA) is a contagious disease of domestic ducks, turkeys and many other birds, also known as the new Duck disease (New duck syndrome, Duck septicaemia, Anatipestfer syndrome, Anatipestifer septicaemia, Pasteurella anatipestifer infection and Duck infectious serosa Infectious serositis. It is a common bacterial infectious disease of ducks, mainly affecting ducklings aged 2-8 weeks, with fibrinous pericarditis, perihepatitis, and air sacculitis as the main pathological features. [0003] Riemerella anatipestifer (RA) is the pathogen that causes Riemerella anatipestifer. This bacterium is a Gram-negative small bacillus, without spores, without pili or flage...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C12N15/13C12N15/70G01N33/569A61K39/395A61P31/04
CPCY02A50/30
Inventor 汪铭书程安春李清竹朱德康刘马峰杨乔
Owner SICHUAN AGRI UNIV
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