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Kit for detecting chicken Marek's disease virus antibody and application thereof

A technique for Marek's disease and Marek's disease in chickens is applied in the field of indirect ELISA kits for detecting antibodies against Marek's disease virus in chickens, and achieves the effects of rapid and sensitive detection, high specificity, and simple and easy experimental operation.

Inactive Publication Date: 2018-01-26
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to overcome the technical defect of existing Marek's disease antibody detection, provide a kind of indirect ELISA kit that utilizes MDV envelope glycoprotein gB protein to detect chicken Marek's disease antibody, and this kit can be used for rapid detection clinical MDV Antibody in Chicken Serum Samples and Antibody Monitoring of Immunized Chickens

Method used

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  • Kit for detecting chicken Marek's disease virus antibody and application thereof
  • Kit for detecting chicken Marek's disease virus antibody and application thereof
  • Kit for detecting chicken Marek's disease virus antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Expression and purification of chicken MDV gB protein of embodiment 1

[0051] 1. Construction and identification of MDV pGEX-6p-1-gB expression vector

[0052] According to the gene sequence of the gB protein published on NCBI (NCBI Reference Sequence: D13713.1), combined with the pGEX-6p-1 vector sequence, the pGEX-6p-1-gB gene was artificially synthesized (synthesized by Wuhan Qingke Innovation Biotechnology Co., Ltd. ), the nucleotide sequence of the gB gene is shown in the sequence listing SEQ ID NO:1.

[0053] Add 2uL of the synthesized plasmid to 50uL DH5α competent cells, and ice-bath for 30min; heat shock at 42°C for 90s and quickly ice-bath for 5min. Add 1mL of antibiotic-free LB liquid medium, mix gently by pipetting, and shake at 37°C for 60min. After the cultured bacterial solution was centrifuged at 1000rpm for 10min, discard the supernatant to about 100uL, gently blow and mix with the precipitate, and then transfer the whole amount to the LB plate conta...

Embodiment 2

[0058] Example 2 Establishment of Chicken MDV Antibody Indirect ELISA Detection Method

[0059] 1. Determination of the optimal reaction conditions of the chicken MDV antibody indirect ELISA detection kit

[0060] The soluble pGEX-6p-1-gB protein prepared by the present invention is used as an antigen coated microtiter plate, and the HRP-labeled goat anti-chicken secondary antibody binds to the chicken serum primary antibody, and catalyzes TMB substrate color development. The optimal coating concentration of the protein was determined to be 1.1ug / mL by square array titration, and the optimal dilution factor of the horseradish peroxidase-labeled antibody was 1:5000.

[0061] 2. Determination of the negative and positive critical values ​​of the chicken MDV antibody indirect ELISA detection kit

[0062] Detect 25 chicken serum samples that are negative for chicken MDV antibody through the agar diffusion test, and set up a standard positive control and a negative control at the ...

Embodiment 3

[0082] Embodiment 3 Assembly of chicken MDV antibody indirect ELISA antibody detection kit

[0083] 1. ELISA kit assembly

[0084] a. 96-well ELISA plate, coated with chicken Marek's disease virus envelope glycoprotein gB protein;

[0085] b. Standard positive control: chicken Marek's disease virus positive serum;

[0086] c. Standard negative control: chicken Marek's disease virus negative serum;

[0087] d. horseradish peroxidase-labeled goat anti-chicken enzyme-labeled antibody;

[0088]e. Sample diluent: 5% ovalbumin solution + 5% horse serum, take 2g of ovalbumin and 5ml of horse serum in 100mL 1×PBS buffer;

[0089] d. Washing solution (20×concentration): by NaCl 160.0g, KH 2 PO 4 5.4g, Na 2 HPO 4 28.4g, KCl 4.0g, Tween20 10mL, add double distilled water to 1000mL to prepare the washing liquid;

[0090] e. Chromogenic substrate solution A: Dissolve 3,3',5,5'-tetramethylbenzidine in DMSO so that the final concentration of 3,3',5,5'-tetramethylbenzidine is 10mg ...

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Abstract

The invention discloses an application of chicken Marek's disease virus envelope glycoprotein gB protein in preparation of an indirect ELISA kit for detecting a chicken Marek's disease virus antibody,and provides the indirect ELISA kit for detecting the chicken Marek's disease virus antibody established by using the MDV envelope glycoprotein gB protein. The kit makes up the detection defects by aiming at the chicken MDV antibody, and has the advantages of simpleness, stabilization and high efficiency, the kit is used for rapidly detecting the MDV virus antibody in a clinical chicken serum sample at an early stage, can be used for monitoring the chicken immune antibody, and is an effective method for large-batch detection, and has the advantages of high specificity, sensitivity, and reappearance. Compared with a current traditional method, the detection effect is better.

Description

technical field [0001] The present invention relates to the field of animal immunology, molecular biology and animal disease detection, more specifically, relates to the application of chicken Marek's disease virus envelope glycoprotein gB protein in the preparation and detection of chicken Marek's disease virus antibody indirect ELISA kit, the present invention The invention also relates to an indirect ELISA kit for detecting chicken Marek's disease virus antibody. Background technique [0002] Marek's disease is a highly contagious, lymphoproliferative disease caused by Marek's disease virus (MDV, hereinafter referred to as MDV). Although vaccines have been used for MD since the 1960s and have played an extremely important role in the prevention of MD, with the enhancement of MDV virulence, MD is still one of the most harmful diseases to the chicken industry today. [0003] The MDV genome is a double-stranded linear DNA with a length of about 175Kb and belongs to the genu...

Claims

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Application Information

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IPC IPC(8): G01N33/535
Inventor 董俊李冉徐高原金梅林陈焕春
Owner WUHAN KEQIAN BIOLOGY CO LTD
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