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Riemerella anatipestifer surface antigen D15 truncated recombinant protein sensitization latex reagent as well as preparation method and application thereof

A technology of Riemer's duck disease and surface antigen, which is applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, can solve the problems of lack of cross-immune protection and difficulty in vaccine prevention and control, and achieves good product agglutination effect. Good reproducibility and sensitivity, high coupling efficiency

Active Publication Date: 2014-07-16
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 sensitization latex reagent as well as preparation method and application thereof
  • Riemerella anatipestifer surface antigen D15 truncated recombinant protein sensitization latex reagent as well as preparation method and application thereof
  • Riemerella anatipestifer surface antigen D15 truncated recombinant protein sensitization latex reagent as well as preparation method and application thereof

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Embodiment 1

[0034] Embodiment 1, the preparation of RA surface antigen D15 truncated recombinant protein

[0035] The amino acid sequence of the RA surface antigen D15 truncated recombinant protein (named tD15) to be prepared in the present invention is shown in SEQ ID No. 1 (i.e. the 94th to 384th amino acid sequence of RA D15 with the accession number AFR36094.1), The nucleotide sequence of the gene encoding it is shown in SEQ ID No. 2 (that is, the 280th to 1152nd nucleotide sequence of the RA D15 gene with the accession number CP003787).

[0036] 1. Cloning of RA tD15 gene

[0037] Design primers according to RA tD15 gene sequence, upstream primer RA tD15-F: 5'-C GGATCC ACAGAATATA-TGACCTACCTAAC-3' (SEQ ID NO.3, the underlined part is the BamHI site); downstream primer RA tD15-R: 5'-CC AAGCTT GGCGCCTATACATCTAAAATAC-3' (SEQ ID NO.4, the underlined part is the HindIII site).

[0038] Extract RA genomic DNA: Inoculate RA CH-1 strain on tryptone soy agar (TSA) plate medium containing ...

Embodiment 2

[0057] Example 2, Preparation and Quality Identification of RA Surface Antigen D15 Truncated Recombinant Protein Sensitization Latex Reagent

[0058] The truncated recombinant protein of RA surface antigen D15 can be used as antigen to prepare sensitized latex reagent, and then detect RA antibody by latex agglutination test.

[0059] 1. Latex agglutination test method and result judgment

[0060] Drop 1 drop (about 20 μL) of the sample solution to be tested on a clean concave glass slide, add 1 drop (about 20 μL) of the sensitizing latex reagent, stir to mix, and shake the slide to form a liquid with a diameter of 1.5 to 2.0 cm. On the surface, observe with naked eyes under a black background for 3 to 5 minutes, and set a positive control and a negative control at the same time. ++++ is 100% latex agglutination, the particles are large and gather at the edge of the droplet, and the liquid is completely transparent; +++ is 75% latex agglutination, the particles are obvious, an...

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Abstract

The invention relates to a riemerella anatipestifer (RA) surface antigen D15 truncated recombinant protein sensitization latex reagent which is latex particle with the surface being coated with RA surface antigen D15 truncated recombinant protein indicated by the amino acid sequence SEQ ID No.1. The sensitization latex reagent can be used for rapidly detecting an RA antibody through a latex agglutination test. The sensitization latex reagent is good in detection specificity, repeatability and sensitivity and can be used for preparing an RA antibody detection reagent and particularly used for preparing an RA antibody latex agglutination detection kit. The invention also discloses a preparation method of the RA surface antigen D15 truncated recombinant protein sensitization latex reagent, the coupling conditions such as the carboxylation latex coupling concentration, coupling concentration and coupling time of the RA surface antigen D15 truncated recombinant protein, and the concentration of closed liquid are searched and optimized, and the preparation method is high in coupling efficiency and good in product agglutination effect.

Description

technical field [0001] The invention belongs to the technical field of bioengineering and detection, and relates to a sensitizing emulsion reagent and its preparation method and application. Background technique [0002] Riemerella anatipestifer (RA) disease is a contagious disease that mainly affects ducks, turkeys and many other birds. Bacillosis and duck infectious serositis are one of the main infectious diseases that endanger the duck industry. The disease was an acute or chronic septic process, and the pathological changes were mainly fibrinous pericarditis, perihepatitis, air sac inflammation, meningitis, and caseous salpingitis. 1-8 week-old ducks are highly sensitive, and harsh environmental conditions or concurrent infections are more likely to cause the occurrence and prevalence of the disease, with high mortality. Durable ducks often grow poorly, gain weight slowly, and lose their feeding value, and the disease can persist in the diseased field, causing differe...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/531C12N15/31C12N15/70C07K14/195
CPCC07K14/195C12N15/70G01N33/531G01N33/559G01N33/56911G01N33/6893
Inventor 程安春奉彬汪铭书朱德康陈孝跃
Owner SICHUAN AGRI UNIV
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