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Kit and application based on recombinant protein of Riemerella anatipestifer omph truncated

A technology of Riemer's duckweed and recombinant protein, applied in the field of bioengineering, can solve the problems of complex bacterial antigen composition, difficult application, low sensitivity, etc., and achieve the effects of wide market application prospect, low production cost and stable performance.

Active Publication Date: 2018-07-10
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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  • Kit and application based on recombinant protein of Riemerella anatipestifer omph truncated
  • Kit and application based on recombinant protein of Riemerella anatipestifer omph truncated
  • Kit and application based on recombinant protein of Riemerella anatipestifer omph truncated

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] The preparation of embodiment 1 Riemerella anatipestifer OmpH truncated recombinant protein

[0057] 1. Main experimental materials

[0058] 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.

[0059] 2 Experimental methods

[0060] 2.1 Cloning of RA OmpH truncated gene

[0061] 2.1.1 Primer design

[0062] According to the existing RA-CH-1OmpH gene sequence in GenBank, the main antigenic domain (81aa-151aa and named as OmpHM) was selected, and a pair of primers were designed using Primer Premier5.0 software. Upstream primer as shown in SEQ ID NO:3: 5'-CGC GGATCC GAGGCTCAGAGAACTGCT-3' (the underlined part is the BamH Ⅰ site); the downstream primer shown ...

Embodiment 2

[0144] Embodiment 2 Purification of Riemerella anatipestifer OmpH truncated recombinant protein

[0145] After the expression product containing the OmpH truncated recombinant protein of Riemerella anatipestifer prepared in Example 1 was collected from the bacteria, ultrasonically disrupted, and supernatant collected, the recombinant protein was purified by nickel agarose gel affinity chromatography. OmpH truncated recombinant 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 50 mmol imidazole, and peak 3 was the elution peak of 100 mmol imidazole. Collect different concentrations of imidazole elution peaks simultaneously, carry out SDS-PAGE electrophoresis, check purity and concentration, the result shows: only contain a large amount of high-purity OmpH truncated recombinant proteins in the 100mmol imidazole elution peak ( Figure 8 ). After the purified OmpH truncated r...

Embodiment 3

[0146] Western-blot (western blot) of embodiment 3 Riemerella anatipestifer OmpH truncated recombinant protein

[0147] 1. Experimental method

[0148] The recombinant protein containing the OmpH truncated fragment of Riemerella anatipestifer obtained in Example 1 was used as a probe for detecting the serum antibody of Riemerella anatipestifer.

[0149] 1SDS-PAGE gel preparation

[0150] 1.1 Preparation of 12% separating gel

[0151] Deionized water 1215ul, 1.5M Tris-HCl (PH=8.8) 950ul, 10% SDS 37.5ul, 10% AP37.5ul, TEMED 1.5ul, 30% acrylamide 1.5ml.

[0152] 1.2 Preparation of 5% stacking gel

[0153] 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.

[0154] 2 Processing of protein samples

[0155] Add an 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...

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Abstract

The invention discloses a kit based on the OmpH truncated recombinant protein of Riemerella anatipestifer and its application, belonging to the field of bioengineering. The ELISA kit of the present invention is composed of a solid phase support, an antibody capture agent, an enzyme-labeled secondary antibody, a substrate, and a blocking solution. The antibody capture agent is an OmpH truncated recombinant protein, and its amino acid sequence is as shown in SEQ ID NO: 1 As shown; the detection method includes: preparation of solid-phase antigen, primary antibody binding, secondary antibody binding, color development, detection and determination; the kit has good specificity, sensitivity and stability; the detection method is simple to operate.

Description

technical field [0001] The invention belongs to the field of bioengineering, and in particular relates to a kit for recombinant OmpH truncated protein of Riemerella anatipestifer and its application. Background technique [0002] Riemerella anatipestifer (RA) is a Gram-negative bacterium belonging to the Flavobacteriaceae family (Flavobacteriaceae), the causative agent of Riemerella anatipestifer, which can cause 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 main clinical symptoms are mental depression, paralysis, neck shrinkage, yellow-green loose stools and other symptoms. The main pathological features of the disease are fibrinous air sacculitis, meningitis, pericarditis, perihepatitis, and caseous salpingitis caused by the host, accompanied by sepsis in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/195
Inventor 汪铭书高群程安春杨乔陈孝跃
Owner SICHUAN AGRI UNIV
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