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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  • Abstract
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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 th

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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[0034] Example 1. Preparation of truncated recombinant protein of RA surface antigen D15

[0035] 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 from position 94 to position 384 of RA D15 with the accession number AFR36094.1), The nucleotide sequence of the coding gene is shown in SEQ ID No. 2 (ie, the nucleotide sequence from position 280 to position 1152 of the RA D15 gene with the accession number CP003787).

[0036] 1. Cloning of RA tD15 gene

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

[0038] Extract RA genomic DNA: inoculate RA CH-1 strain in tryptone soy agar (TSA) plate medium contai...

Example Embodiment

[0057] Example 2. Preparation and quality identification of RA surface antigen D15 truncated recombinant protein sensitized latex reagent

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

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

[0060] Drop 1 drop (about 20μL) of the sample solution to be tested on a clean concave glass slide, add 1 drop (about 20μL) of sensitizing latex reagent, stir and mix, shake the slide to form a liquid with a diameter of 1.5~2.0cm Surface, 3~5min built-in black background for naked observation, and set positive control and negative control at the same time. ++++ means 100% latex agglutination, the particles are large and gathered at the edge of the droplet, the liquid is completely transparent; +++ means 75% latex agglomerates, the particles are obvious, the liquid is slightly turbid; ++ means 50% ...

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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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