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Antigen for detecting sheep echinococcus antibody and preparation method of agar diffusion plate

An antibody and antigen technology, which is applied in the field of antigen detection for echinococcosis antibody and the preparation of agar diffusion plate, to achieve the effects of clear precipitation line, good biological safety and strong specificity

Pending Publication Date: 2020-11-03
CHONGQING AULEON BIOLOGICALS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Ordinary agar diffusion plate formula, the detection sensitivity of using a single antigen is not as good as that of encapsulation fluid and other antigens, and the sensitivity needs to be improved

Method used

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  • Antigen for detecting sheep echinococcus antibody and preparation method of agar diffusion plate
  • Antigen for detecting sheep echinococcus antibody and preparation method of agar diffusion plate
  • Antigen for detecting sheep echinococcus antibody and preparation method of agar diffusion plate

Examples

Experimental program
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Effect test

Embodiment 1

[0052] In this embodiment, the preparation method of the antigen for detecting sheep echinococcosis antibody comprises the following steps:

[0053] 1) Amplify the EG95 gene fragment by PCR to obtain the nucleic acid sequence shown in SEQ ID NO: 1;

[0054] 2) The nucleic acid sequence is digested simultaneously with EcoR I and Xho I, and connected to the pET32a plasmid vector to construct an expression plasmid;

[0055] 3) Transform the expression plasmid into Escherichia coli BL21(DE3), construct the expression strain, culture the expression strain and perform IPTG-induced expression to obtain the fermentation broth; specifically: the glycerol strain, at a ratio of 2% (V / V) Insert 200mL of LB liquid medium containing Amp (100μg / ml), and culture at 37°C with shaking at 150r / min until the OD600nm reaches about 0.5 to obtain the primary fermentation liquid; transfer the primary fermentation liquid to 2% Insert 5L of LB liquid medium containing Amp (100μg / mL), 150r / min, 37°C sh...

Embodiment 2

[0070] In this example, the preparation method of the agar diffusion plate for detecting the antigen of sheep Echinococcus antibody comprises the following steps: add 90 mL of deionized water and 10 mL of 0.1 mM PBS to 0.8 g of agarose, and heat the agarose on a magnetic stirrer to Melt completely, stop heating, then add polyethylene glycol 6000 1g, MES 1g and sodium chloride 0.8g, stir continuously until completely melted, cool to 45-60°C and pour into a plate with a plate thickness of about 3.5mm. Use a hexagonal puncher to punch holes on the agar plate according to the 7-hole plum blossom pattern, with a hole diameter of about 5mm and a hole distance of 3mm, and use a syringe needle to pick out the agar in the holes; after punching, bake the bottom of the plate on an alcohol lamp to cover , to slightly melt the bottom agar.

Embodiment 3

[0072] Establishment of the agar diffusion method

[0073] 1. Preparation of experimental serum:

[0074] Positive control serum: select 3 healthy goats and sheep of 2 to 4 months old from non-infected areas and non-immunized, and dilute the genetically engineered subunit vaccine of echinococcosis (hydatid) disease with a special diluent according to the instructions, and then pass Inject immunized lambs subcutaneously in the neck, 1 dose per lamb, and boost immunization once 28 days later with the same dose and route. 14 days after the second immunization, aseptically collect blood via the jugular vein, routinely separate the serum, and store it at -20°C for later use;

[0075]Negative control serum: 3 non-immune healthy goats and 3 sheep from non-epidemic areas, 2 to 4 months old, blood was collected via jugular vein aseptically, serum was routinely separated, and stored at -20°C for later use. The other specific experimental sera were prepared according to the normal immu...

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Abstract

The invention discloses an antigen for detecting a sheep echinococcosis antibody and a preparation method of an agar diffusion plate, and belongs to the technical field of biological detection. The preparation method of the antigen comprises the following steps: performing enzyme digestion connection on a sheep echinococcosis EG95 protein nucleic acid sequence, and constructing the sheep echinococcosis EG95 protein nucleic acid sequence on a plasmid vector; then performing converting into escherichia coli, constructing an expression strain, performing IPTG induced expression, and performing crushing to obtain an inclusion body; and performing washing, cracking, centrifuging, purifying, dialyzing, concentrating, freeze-drying, and diluting with PBS. The preparation method of the agar diffusion plate comprises the following steps: adding water and PBS into agarose, performing heating, adding polyethylene glycol 6000, MES and sodium chloride, performing cooling, performing punching, and sealing the bottom. The antigen and the agar diffusion plate provided by the invention have the advantages of high sensitivity, strong specificity and strong universality, have good biosafety, are easyfor large-scale production, are especially suitable for clinical detection of sheep serum by grassroots veterinarians, and have wide popularization and application value.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a method for preparing an antigen for detecting sheep echinococcosis antibody and an agar diffusion plate. Background technique [0002] Echinococcus granulosus (Echinococcus granulosus, Eg) larvae infecting sheep is a zoonotic disease, commonly known as hydatidosis. It is a zoonotic parasitic disease that seriously endangers health and is caused by the continuation larvae of Echinococcus granulosus parasitic in human liver, lung and other organs. It is one of the five major parasitic diseases planned by the Chinese health department. . Echinococcosis is mainly prevalent in 350 counties in the agricultural and pastoral areas of western my country, with a threatened population of about 50 million. The average prevalence of echinococcosis in plateau areas is 1.20%, and in some cases it is as high as 12%. According to the calculation of the agricultural de...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/70C07K1/36C07K1/34C07K1/22G01N33/68
CPCC07K14/43554C12N15/70G01N33/6854G01N33/6893G01N2333/43543
Inventor 赵扬扬赖茂林王盼举伏刚冉智光吕航古静殷远滔杨元礼
Owner CHONGQING AULEON BIOLOGICALS
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