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Riemerella anatipestifer OmpA/MotB signal peptide-removal recombinant protein, antibody and preparation method and application thereof

A technology of Riemerella anatipestifer and de-signal peptide is applied in the field of genetic engineering to achieve the effect of simple preparation method and standardization

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 thrombin type 3 repeat protein (Thrombospondin type 3 repeats): OmpA / MotB gene and its protein of Riemerella anatipestifer at home and abroad, therefore, Thrombospondin type 3 repeats: OmpA / MotB gene and its protein The research helps to open up new ideas for the prevention and control of RA

Method used

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  • Riemerella anatipestifer OmpA/MotB signal peptide-removal recombinant protein, antibody and preparation method and application thereof
  • Riemerella anatipestifer OmpA/MotB signal peptide-removal recombinant protein, antibody and preparation method and application thereof
  • Riemerella anatipestifer OmpA/MotB signal peptide-removal recombinant protein, antibody and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of recombinant protein without signal peptide OmpA / MotB

[0032] 1. Primer design for OmpA / MotB gene (without signal peptide)

[0033] According to the RA-CH-1 bioinformatics analysis and the predicted OmpA / MotB gene sequence (Genbank ID number: CP003787.1) information published by Gene Bank, the OmpA / MotB gene fragment is 1467bp in length, and SignalP 4.1Server is used for signal peptide prediction. Bases 1-63 of the OmpA / MotB gene are signal peptide sequences. Use the Primer Premier5.0 software to design primers for the conserved region that removes the signal peptide as follows, through the amplification of the primers, the length of the primer region of the OmpA / MotB gene without the signal peptide fragment is 1563bp, plus the enzyme cleavage site and protective base The size of the post-basic PCR product is 1581bp. The nucleotide sequence of the OmpA / MotB de-signal peptide expression product is shown in SEQ ID NO.1, and the amino acid sequenc...

Embodiment 2

[0059] Example 2 Establishment of an indirect ELISA method based on OmpA / MotB without signal peptide protein

[0060] 1. Optimization of reaction conditions based on OmpA / MotB de-signal peptide protein ELISA method

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

[0062] Table 1 Optimization of indirect ELISA conditions

[0063]

[0064] Optimize according to the following steps with each reaction condition of table 1:

[0065] (1) Dilute the protein with coating solution, add 100 μL to each well of the microplate, and overnight at 4°C;

[0066] (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;

[0067] (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;

[0068] (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;

[0069] (5) W...

Embodiment 3

[0122] Example 3 Preparation and application of rabbit anti-OmpA / MotB de-signal peptide recombinant protein hyperimmune serum

[0123] 1. Preparation of immunogen

[0124] The OmpA / MotB signal peptide-removed 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 emulsified by ultrasonication to form oil Until the water-containing agent. 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.

[0125] 2. Animal immunity

[0126] 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.

[0127] Table 16 Preparation of hyperimmune serum of rabbit anti-OmpA / MotB de-signal peptide recombinant pro...

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Abstract

The invention discloses a Riemerella anatipestifer OmpA / MotB signal peptide-removal 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 recombinant protein of Riemerella anatipestifer OmpA / MotB without signal peptide, an antibody thereof, a preparation method and application thereof. 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 spo...

Claims

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Application Information

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IPC IPC(8): C07K16/18C12N15/13C12N15/70C12N1/21G01N33/569A61K39/395A61P31/04
CPCY02A50/30
Inventor 程安春汪铭书李清竹杨乔
Owner SICHUAN AGRI UNIV
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