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Preparation method and application kit of rabbit anti-polyclonal antibody specifically recognizing phytoplasma jujube

The technology of polyclonal antibody and phytoplasma is applied in the field of preparation of rabbit anti-polyclonal antibody, which can solve the problems of high detection cost and cumbersome detection process of jujube madness, achieve remarkable specificity, prevent the spread of jujube madness, guarantee The effect of farmers

Active Publication Date: 2020-06-02
INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Aiming at the above-mentioned technical problems such as cumbersome detection process of jujube madness, high detection cost, and difficult detection technology, the present invention proposes a specific identification method with reasonable design, simple method, convenient operation and no need for professional knowledge to detect jujube madness Preparation method of rabbit anti-polyclonal antibody against phytoplasma jujube and its application kit

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  • Preparation method and application kit of rabbit anti-polyclonal antibody specifically recognizing phytoplasma jujube
  • Preparation method and application kit of rabbit anti-polyclonal antibody specifically recognizing phytoplasma jujube
  • Preparation method and application kit of rabbit anti-polyclonal antibody specifically recognizing phytoplasma jujube

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Embodiment 1

[0038] Example 1, a rabbit anti-polyclonal antibody that specifically recognizes phytoplasma jujube madness for the detection of jujube madness in this embodiment

[0039] In order to produce antibodies against the phytoplasma immune membrane protein of jujube mad disease, it is first necessary to collect pathogenic materials. In this example, go to the jujube tree planting area to collect jujube mad disease samples showing arbuscular and leaflet symptoms, and sequence through 16Sr RNA, rp and tuf genes It shows that the sample does contain Phytoplasma jujube, which provides a good pathogenic basis for the subsequent sequence of Phytoplasma jujube.

[0040]In order to obtain a large number of gene sequences of the immune membrane protein of Phytoplasma japonica, primer pairs were designed according to the conserved regions on both sides of the gene sequence of the phytoplasma immune membrane protein, and the PCR amplification of the immune membrane protein of the phytoplasma ph...

Embodiment 2

[0080] Example 2, considering that centrifugation requires certain professional knowledge and equipment, for this reason, this example also provides another specific operation procedure for preparing a kit for the above-mentioned rabbit anti-polyclonal antibody that specifically recognizes Phytoplasma japonica is as follows :

[0081] A. Sample processing: Take 0.05g ~ 0.10g of suspected disease-like fresh branches into a centrifuge tube, then add 2-5 small steel balls and 0.5-1ml antigen coating buffer, grind with a grinding rod until the sample is paste, Let stand for 15 minutes, take the supernatant and dilute 2 times with antigen coating buffer for use.

[0082] B. Sample coating: Take 150 μL of the diluted test sample solution and add it to a 96-well microtiter plate, use the positive standard of Phytoplasma jujube as the positive control, and the healthy jujube standard as the negative control, and incubate overnight at 4°C Or incubate at 37°C for 1-2h;

[0083]C. Wash...

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Abstract

The invention belongs to the field of detection of jujube witches broom phytoplasma and in particular relates to a preparation method and application kit of a rabbit anti-polyclonal antibody for specifically identifying the jujube witches broom phytoplasma. The rabbit anti-polyclonal antibody for specifically identifying the jujube witches broom phytoplasma, provided by the invention, is mainly used for detecting jujube witches broom; the preparation method of the rabbit anti-polyclonal antibody comprises the following effective steps: a, cloning from total DNA (Deoxyribonucleic Acid) of a jujube tree infected by the jujube witches broom phytoplasm to obtain an immunodominant membrane protein gene sequence of the jujube witches broom phytoplasm and predicating an amino acid sequence of immunodominant membrane protein; selecting a 47-152aa region with relatively good C-terminal epitope exposure and carrying out protein expression; then carrying out label purification; b, after the labelpurification is carried out, mixing the selected protein and an incomplete Freund's adjuvant and immunizing Japanese white rabbits; collecting blood of the rabbits; carrying out antigen affinity purification to obtain a polyclonal antibody of the jujube witches broom phytoplasma immunodominant membrane protein. The rabbit anti-polyclonal antibody for specifically identifying the jujube witches broom phytoplasma, provided by the invention, has remarkable specificity in the detection of the jujube witches broom and the detection accuracy is ensured.

Description

technical field [0001] The invention belongs to the field of detection of phytoplasma jujube, in particular to a preparation method of a rabbit anti-polyclonal antibody for specifically recognizing phytoplasma jujube and an application kit thereof. Background technique [0002] Jujube tree is native to my country, and its cultivated varieties and wild jujube resources are extremely rich. It is one of the characteristic fruit tree species with important economic value in my country. Jujube madness is the most serious disease of jujube trees in my country, and it is also one of the typical phytoplasma diseases in the world. [0003] Phytoplasma is a kind of pathogenic bacteria without cell walls that exists in plant sieve tubes. It mainly relies on leafhoppers and other Hemiptera insects that feed on the phloem, and can also be transmitted by dodder and grafting. Therefore, phytoplasma membrane proteins may directly interact with Certain components of insect or plant cytoplas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/535C12N15/31C07K16/12C07K16/06
CPCC07K14/195C07K16/12C07K2317/10G01N33/535G01N33/56911G01N2333/195G01N2333/415
Inventor 高瑞王洁杨淑珂李凡路兴波
Owner INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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