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Riemerella anatipestifer OmpH recombinant protein and ELISA kit thereof

A Riemerella anatipestifer, recombinant protein technology, applied in the field of bioengineering, can solve the problems of unrecognized Riemerella anatipestifer OmpH research and application research reports, complex bacterial antigen components, and high bacterial culture requirements, reaching the market. Wide application prospects, maintaining natural activity, and high sensitivity

Active Publication Date: 2015-10-21
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, agar amplification test and agglutination test have low sensitivity and low specificity, and can only detect individual serum; although PCR detection is sensitive, it is difficult to use in actual production and requires professional instruments and personnel; the ELISA method of crushing whole bacteria Although it has demonstrated the advantages in ELISA detection, as a coating antigen, the requirements for bacterial culture are high when preparing bacterial antigens, and there are deficiencies in the complex components and specificity of bacterial antigens.
[0004] OmpH belongs to the porin, and there is no relevant research report on the research and application of OmpH of Riemerella anatipestifer

Method used

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  • Riemerella anatipestifer OmpH recombinant protein and ELISA kit thereof
  • Riemerella anatipestifer OmpH recombinant protein and ELISA kit thereof
  • Riemerella anatipestifer OmpH recombinant protein and ELISA kit thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of Riemerella anatipestifer OmpH recombinant protein

[0052] 1. Main experimental materials

[0053] Plasmid T-Vector pMD19(simple), Max DNA Polymerase was purchased from Dalian Bao Biological Engineering Co., Ltd.; prokaryotic expression plasmid pET32a(+), product of Novagen; clone host bacteria E.coli DH5α, expression host bacteria E.coli BL21(DE3) and RA-CH-1 The strains were provided by the Poultry Disease Research Center of Sichuan Agricultural University.

[0054] 2 Experimental methods

[0055] 2.1 Cloning of RA OmpH gene

[0056] 2.1.1 Primer design

[0057] According to the RA-CH-1 OmpH gene sequence on GenBank, a pair of primers were designed using Primer Premier5.0 software. Upstream primer as shown in SEQ ID NO:3: 5'- GGATCC ATGAAAAAATTAAGCGTATTGTTTGC-3' (the underlined part is the BamHI site); the downstream primer shown in SEQ ID NO: 4: 5'- CTCGAG ATTTTACATTATTGAGATGCCCTATTG-3' (the underlined part is the XhoI site). After...

Embodiment 2

[0138] Example 2 Purification of Riemerella anatipestifer OmpH recombinant protein

[0139] After the expression product containing the OmpH recombinant protein of Riemerella anatipestifer prepared in Example 1 was processed through a series of treatments such as collecting the bacterium, ultrasonic crushing, and collecting the supernatant, the recombinant OmpH recombinant protein was purified by nickel agarose gel affinity chromatography. protein. The UV curve of the protein sample after column purification showed three peaks, peak 1 was the breakthrough peak, peak 2 was the elution peak of 200 mmol imidazole, and peak 3 was the elution peak of 250 mmol imidazole. Collect different concentration imidazole elution peaks simultaneously, carry out SDS-PAGE electrophoresis, check purity and concentration, the result shows: in 250mmol imidazole elution peaks, contain a large amount of high-purity OmpH recombinant protein ( Figure 8 ). After the purified OmpH recombinant protein...

Embodiment 3

[0140] Example 3 Western-blot (western blot) of Riemerella anatipestifer OmpH recombinant protein

[0141] 1. Experimental method

[0142] The recombinant protein containing R. anatipestifer OmpH obtained in Example 1 was used as a probe for detecting serum antibodies to R. anatipestifer.

[0143] 1 SDS-PAGE gel preparation

[0144] 1.1 Preparation of 12% separating gel

[0145] Deionized water 1215ul, 1.5M Tris-HCl (PH=8.8) 950ul, 10% SDS 37.5ul, 10% AP 37.5ul, TEMED 1.5ul, 30% acrylamide 1.5ml.

[0146] 1.2 Preparation of 5% stacking gel

[0147] Deionized water 700ul, 1.0M Tris-HCl (PH=6.8) 125ul, 10% SDS 10.0ul, 10% AP 10.0ul, TEMED 1.0ul, 30% acrylamide 165ul.

[0148] 2 Processing of protein samples

[0149] Add appropriate amount of SDS-PAGE protein loading buffer (0.5M Tris-HCl (PH=6.81.2ml), glycerin 1ml, deionized water 4.8ml, 10% SDS 2.0ml, 0.1% BPB 0.5ml) to the protein sample . 10000r / min 10min after boiling at 100℃ for 10min.

[0150] 3 Electrophoresis

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Abstract

The invention discloses a riemerella anatipestifer OmpH recombinant protein and an ELISA kit thereof and belongs to the field of bioengineering. The ELISA kit is composed of a solid-phase supporting object, an antibody trapping agent, bethyl, a substrate and confining liquid, wherein the antibody trapping agent is the riemerella anatipestifer OmpH recombinant protein, and the amino acid sequence of the riemerella anatipestifer OmpH recombinant protein is shown in SEQ ID NO:1. A detecting method comprises the steps of preparation of solid-phase antigen, primary antibody combination, secondary antibody combination, color development, detection and determination. The kit is good in specificity, sensitivity and stability, and the detecting method is simple in operation.

Description

technical field [0001] The invention belongs to the field of biological engineering, in particular to a Riemerella anatipestifer OmpH recombinant protein and an ELISA kit thereof. Background technique [0002] Riemerella anatipestifer (RA) is a Gram-negative bacterium belonging to the family Flavobacteriaceae and is the causative agent of Riemerella anatipestifer disease in ducks, geese, turkeys and many It is a chronic or acute septic contagious disease of poultry species, which is distributed worldwide, with high morbidity and mortality, and has become one of the main infectious diseases that seriously endanger the duck industry. The clinical symptoms of the disease mainly manifested as depression, paralysis, neck constriction, yellow-green loose stools and other symptoms; the pathological changes were characterized by fibrinous air sac inflammation, meningitis, pericarditis, perihepatic inflammation, and caseous fallopian tubes caused by the host. Inflammation, accompani...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/31G01N33/68G01N33/569
CPCC07K14/195G01N33/56911G01N33/68G01N2333/195
Inventor 程安春高群汪铭书朱德康杨乔陈孝跃
Owner SICHUAN AGRI UNIV
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