Riemerella anatipestifer OmpA/MotB truncated recombinant protein, antibody and preparation method and application thereof

A technology of Riemeria anatipestifer and recombinant protein is applied in the field of genetic engineering to achieve standardization and simple preparation methods

Active Publication Date: 2015-10-14
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Since there is no report on the research on Thrombospondin type 3 repeats (Thrombospondin type 3 repeats): OmpA/MotB gene and its protein of Riemeria anatipestifer at

Method used

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  • Riemerella anatipestifer OmpA/MotB truncated recombinant protein, antibody and preparation method and application thereof
  • Riemerella anatipestifer OmpA/MotB truncated recombinant protein, antibody and preparation method and application thereof
  • Riemerella anatipestifer OmpA/MotB truncated recombinant protein, antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

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

[0033] According to the bioinformatics analysis of RA-CH-1 published by Gene Bank and the predicted OmpA / MotB gene sequence (Genbank ID number: CP003787.1), the OmpA / MotB gene fragment is 1467bp long. Bit analysis, further use the Protein in DNAstar7.0 to carry out antigen analysis on the sequence, and select the main epitope segment of RA-CH-1 OmpA / MotB: 409bp-1389bp segment (969bp) for truncated expression. Using the extracted RA-CH-1 genomic DNA as a template, amplified by PCR, the DNA fragment of the main antigenic epitope region of the OmpA / MotB gene was obtained, the expected length (primer region plus restriction site) 981bp, 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.

[0034] PCR cloning primers:

[0035] Upstream prime...

Embodiment 2

[0059] Example 2 Establishment of an 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 optimized conditions are shown in Table 1.

[0062] Table 1 Optimization of indirect ELISA conditions

[0063]

[0064]

[0065] Follow the steps below to optimize each reaction condition in Table 1:

[0066] (1) Dilute the protein with the coating solution, add 100 μL to each well of the microplate, 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 h;

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

[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 h;

[0070] (5) Wash the plate as above, pat dry, add 100 ...

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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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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