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Riemerella anatipestifer surface antigen d15 truncated recombinant protein and its preparation method and application

A duck plague Rimmer's and surface antigen technology, applied in the field of truncated recombinant protein and its preparation, can solve the problems of vaccine prevention and control, lack of cross-immune protection, etc., achieve standardization, simple preparation method, and facilitate the comparison of results

Active Publication Date: 2015-09-23
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the many and complex serotypes of RA, there are 21 serotypes recognized internationally, and there are more than 10 serotypes in my country, and there is a lack of effective cross-immune protection between the serotypes, which brings certain difficulties to the vaccine prevention and treatment of RA.

Method used

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  • Riemerella anatipestifer surface antigen d15 truncated recombinant protein and its preparation method and application
  • Riemerella anatipestifer surface antigen d15 truncated recombinant protein and its preparation method and application
  • Riemerella anatipestifer surface antigen d15 truncated recombinant protein and its preparation method and application

Examples

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Effect test

Embodiment 1

[0030] Example 1. Preparation of truncated recombinant protein of RA surface antigen D15

[0031] The amino acid sequence of the RA surface antigen D15 truncated recombinant protein (named tD15) to be prepared by the present invention is shown in SEQ ID No. 1 (that is, the amino acid sequence of RA D15 from 94th to 384th with the accession number AFR36094.1) The nucleotide sequence of the coding gene is shown in SEQ ID No. 2 (that is, the nucleotide sequence from position 280 to position 1152 of the RA D15 gene with accession number CP003787).

[0032] 1. Cloning of tD15 gene

[0033] Design primers based on tD15 gene sequence, upstream primer RA tD15-F: 5’-C GGATCC ACAGAATATATGA-CCTACCCTAAC-3' (SEQ ID NO. 3, underlined part is BamH Ⅰ site); downstream primer RA tD15-R: 5'-CC AAGCTT GGCGCCTATACATCTAAAATAC-3' (SEQ ID NO. 4, the underlined part is HindIII site).

[0034] Extract RA genomic DNA: inoculate RA CH-1 strain in tryptone soy agar (TSA) plate medium containing 5% bovine serum...

Embodiment 2

[0053] Example 2. Application of RA surface antigen D15 truncated recombinant protein in preparation of RA antibody detection reagent

[0054] The RA surface antigen D15 truncated recombinant protein can be used as a coating antigen to detect RA antibodies by indirect ELISA.

[0055] 1. Indirect ELISA method

[0056] Dilute the purified recombinant tD15 / His-tag fusion protein with the coating solution and add it to the ELISA plate, 100μL / well, and coat overnight at 4°C; discard the liquid, wash three times, add 200μL blocking solution to each well, and block at 37°C for 30min ; Discard the liquid, wash three times, add 100μL of serum to be tested diluted with antibody diluent to each well, incubate at 37°C for 30 minutes; discard the liquid, wash three times, add 100μL of diluted goat anti-duck IgG-HRP enzyme-labeled secondary antibody to each well ( (Purchased from KPL Company, USA), incubate at 37°C for 30 minutes; discard the liquid, wash three times, add 100μL of tetramethylbenz...

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Abstract

The invention discloses an RA (Riemerella Anatipestifer) surface antigen D15 truncated recombinant protein as shown in an amino acid sequence, namely SEQ ID No.1, a coding gene, a recombinant expression vector and an engineering bacterium thereof and a method for preparing the truncated recombinant protein by utilizing the engineering bacterium. The truncated recombinant protein disclosed by the invention is similar to a natural D15 protein in immune reaction activity, can be specifically combined with an RA antibody, can not generate the cross reaction with the positive serums of other 6 common toxic strains or strains, can be applied as a detection antigen used for detecting the RA whole bacteria antibody and generates no toxin dispersing danger when being used for detection due to non-whole bacteria. The preparation method of the truncated recombinant protein, which is disclosed by the invention, is simple and suitable for industrialized large-scale production, can realize the standardization of products and is favorable to the result comparison of different laboratories.

Description

Technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a truncated recombinant protein and a preparation method and application thereof. Background technique [0002] Riemerella Anatipestifer (RA) disease is a contagious disease that mainly affects ducks, turkeys and a variety of other birds. It is also called new duck disease, duck disease syndrome, duck disease pasteurella Bacillosis and infectious serositis of ducks are one of the main infectious diseases that currently endanger the duck industry. The disease presents an acute or chronic septicemia process, and the pathological changes are mainly fibrinous pericarditis, perhepatitis, air sacculitis, meningitis, and caseous salpingitis. Ducks of 1-8 weeks old are highly sensitive, and harsh environmental conditions or concurrent infections are more likely to cause the occurrence and epidemic of the disease, and the mortality rate is high. Over-tolerant ducks often grow poorly, ga...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/195C07K1/36C07K1/22C07K1/34C12N15/31C12N15/70C12N1/21G01N33/68
CPCC07K14/195C12N15/70G01N33/56911
Inventor 程安春奉彬汪铭书朱德康陈孝跃
Owner SICHUAN AGRI UNIV
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