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Kit based on duck plague virus gG segmented recombinant protein and its application

A duck plague virus and recombinant protein technology, applied in the field of bioengineering, can solve the problems of cumbersome methods, false positive results, time-consuming and labor-intensive problems, and achieve the effects of easy purification, stable performance and low production cost

Active Publication Date: 2012-02-22
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the past, the detection method of serum antibody to duck plague virus mainly used the whole virus as the antigen for detection, but the traditional virus isolation and identification method takes about 4 to 5 days, the method is cumbersome, time-consuming and laborious, and the purified virus will be mixed with Numerous host cell components, leading to false positive results

Method used

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  • Kit based on duck plague virus gG segmented recombinant protein and its application
  • Kit based on duck plague virus gG segmented recombinant protein and its application
  • Kit based on duck plague virus gG segmented recombinant protein and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Preparation of duck plague virus gG truncated recombinant protein

[0050] One material preparation

[0051] 1 Strains, plasmids and strains

[0052] Plasmid pMD18-T, 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 virulent strain of DPV CHv , provided by the Poultry Disease Research Center of Sichuan Agricultural University. Both goat anti-duck IgG-HRP (horseradish peroxidase-labeled goat anti-duck IgG) and tetramethylbenzidine (TMB) were purchased from KPL in the United States. 0.1mg / bottle, dissolve to 1mL according to the instructions.

[0053] 2 Test animals

[0054] The 10-day-old duck embryo was negative for DPV and antibody of the breeding duck.

[0055] 3 Experimental methods

[0056] 3.1 Cloning of DPV gG gene

[0057] 3.1.1 Primer design

[0058] According to the main antigen domain (68aa-...

Embodiment 2

[0136] Example 2 Purification of duck plague virus gG truncated recombinant protein

[0137] After the expression product containing the gG truncated recombinant protein of duck plague virus obtained in Example 1 was subjected to a series of pretreatments such as lysozyme lysis, ultrasonication, and urea washing, the recombinant gG protein was purified by nickel agarose gel affinity chromatography. 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 300 mmol imidazole. At the same time, the elution peaks of different concentrations of imidazole were collected, and SDS-PAGE electrophoresis was performed to check the purity and concentration. The results showed that only the elution peak of 300mmol imidazole contained a large amount of high-purity gG truncated recombinant protein ( Figure 8 ). After dialysis and renaturation of t...

Embodiment 3

[0139] Example 3 Duck plague virus gG truncated recombinant protein as an indirect ELISA kit for duck plague virus antibody capture agent

[0140] The application of duck plague virus gG truncated recombinant protein as a duck plague virus antibody capture agent is mainly explained through the indirect ELISA kit.

[0141] Solid phase carrier: The solid phase carrier is used as an adsorbent and container during the ELISA determination process, and does not participate in chemical reactions. There are many materials that can be used as solid-phase carriers in ELISA, such as polystyrene, which must meet the requirements of strong protein adsorption properties. After antibodies or protein antigens are adsorbed on it, they still retain their original immunological activity and can be made into various forms. . There are three main shapes of ELISA carriers: microtiter plates, beads, and small test tubes. The microtiter plate is the most commonly used, and the product dedicated t...

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Abstract

Relating to the field of biological engineering, the invention especially relates to a kit for detecting a duck plague virus antibody and a detection method thereof. The ELISA kit comprises a solid support, an antibody scavenger, an enzyme-labeled secondary antibody, a substrate and a blocking solution. The antibody scavenger is the duck plague virus gG segmented recombinant protein, which has anamino acid sequence as shown in SEQ ID NO: 1. The detection method consists of: solid phase antigen preparation, first antibody combination, secondary antibody combination, color development, detection and determination. The kit of the invention has the advantages of good specificity, sensitivity and stability. And the detection method provided in the invention is simple to operate.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a kit and a detection method for detecting duck plague virus antibodies. Background technique [0002] Duck plague (Duck Plague, DP) is an acute, febrile, contact-infected septicemia infectious disease of ducks, geese, swans and other waterfowl caused by duck plague virus (DPV) in the herpesviridae family . The disease can lead to a decrease in egg production and death of commercial waterfowl, and it also has a different lethality rate to wild waterfowl. The clinical manifestations of DP are mental depression, persistent high fever, numbness of legs, diarrhea, tearing, and swelling of the head and neck of some sick ducks. It is an infectious disease that is widely addicted to systemic infection. Vascular injury, tissue and organ bleeding, gastrointestinal bleeding, inflammation and necrosis, rash-like damage in certain parts of the gastrointestinal mucosa, lymphoid organ damage, a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/535G01N33/569C07K14/03
Inventor 程安春杨晓圆汪铭书陈孝跃
Owner SICHUAN AGRI UNIV
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