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Fusion antigen for detecting echinococcosis, coding gene thereof, host cell and kit

A fusion antigen, echinococcosis technology, applied in the detection of echinococcosis fusion antigen, host cells and kits, and its coding gene field, can solve the problems of specificity and sensitivity improvement degree limitation, to improve sensitivity and specificity sexual effect

Active Publication Date: 2019-05-17
青海省地方病预防控制所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] In summary, although there are many types of antigens used to detect echinococcosis, a single antigen or a combination of different antigens is generally used in the detection, and the specificity and sensitivity of the detection are limited to a certain extent.

Method used

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  • Fusion antigen for detecting echinococcosis, coding gene thereof, host cell and kit
  • Fusion antigen for detecting echinococcosis, coding gene thereof, host cell and kit
  • Fusion antigen for detecting echinococcosis, coding gene thereof, host cell and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] This example is a preparation example of the fusion antigen.

[0049] 1. Synthesize the gene of the fusion antigen, its sequence is shown in SEQ ID NO:6. Amplify the reaction using the following reaction system:

[0050] 50ul system

[0051]

[0052] in:

[0053] The sequence of primer 1 is: CGCGGATCCATGAAAGATGAGCCAAAAGCA, which contains a BamHI restriction site;

[0054] The sequence of primer 2 is: CCCTCGAGTTATTTGAGGTTGGCCAGCT, which contains an XhoI restriction site.

[0055] The PCR amplification conditions are:

[0056] 35 cycles of reaction process:

[0057] 94°C: 7min

[0058] 94°C: 30s

[0059] 60°C: 30s

[0060] 72°C: 2min

[0061] 72°C: 10min

[0062] 4°C: for eve.

[0063] 2. Identification by double enzyme digestion

[0064] The reaction system and conditions for double enzyme digestion identification are as follows:

[0065] System 40ul:

[0066]

[0067] React at 37°C for two hours.

[0068] 3. Activation of plasmid glycerol bacteria: ...

Embodiment 2

[0156] This embodiment is an example of the preparation and use of the reagent strip. Specifically, the detection area (T line) on the NC membrane of the test card is coated with the echinococcosis antigen of Example 1, and the quality control area (C line) is coated with anti-hydatid antibody.

[0157] (1) Coating: Dilute the fusion antigen of Example 1 to 0.2 mg / ml as a T-line coating solution, and dilute the anti-hydatid antibody to 2 mg / ml as a C-line coating solution. Coat the T-line coating solution and the C-line coating solution on the nitrocellulose membrane through a film spraying machine. to dry.

[0158] (2) UCP-labeled antigen: The UCP-labeled antigen in this kit is obtained through the following steps:

[0159] a) Weigh 10 mg of UCP particles and place them in a conical flask;

[0160] b) Add 10ml pH=7.2 0.20M PB;

[0161] c) Add 0.5 mg of the antigen of Example 1 to the UCP particle suspension, then add anhydrous glutaraldehyde to a final concentration of 1%...

Embodiment 3

[0176] This embodiment is an application example of the reagent strip. During the test, the sample is added to the sampling point of the test card, and the liquid is chromatographed under the capillary effect. The substance will be bound by the antigen coated on the T line, and a solid-phase UCP particle-echinococcosis marker antigen-echinococcosis antibody-echinococcosis antigen complex will be formed at the position of the T line, and a solid-phase complex will be formed at the C line Anti-hydatid antibody-labeled antigen-UCP complex. Finally, the detection result is obtained by detecting the emitted light signal of the upconverted luminescent particle.

[0177] Table 1 - Echinococcosis serum antibody detection results

[0178]

[0179] Comparative detection of echinococcosis serum antibody detection reagents and commercially available reagents:

[0180] Comprehensive evaluation of 108 cases of echinococcosis positive samples (ELISA) and 162 cases of normal human serum...

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Abstract

The present invention discloses a fusion antigen for detecting echinococcosis, a coding gene thereof, a host cell and a kit. The fusion antigen for detecting echinococcosis includes a cystic hydatid antigen portion A, a vesicular hydatid antigen portion B, and a spacer sequence between the antigen portion A and the antigen portion B. The antigen portion A includes multiple epitopes and located atan N-end of the fusion antigen, the antigen portion B is a single epitope and is located at a C-end of the fusion antigen, the spacer sequence chemically links the antigen portion A with the antigen portion B to achieve fusion. The fusion antigen has higher sensitivity and specificity.

Description

technical field [0001] The invention relates to the detection of echinococcosis, in particular to a fusion antigen for detecting echinococcosis, its coding gene, host cell and kit. Background technique [0002] Hydatidosis (hydatid disease) is a parasitic disease caused by the larval infection of Echinococcus tapeworm. The final hosts of Echinococcus granulosus are dogs, wolves, foxes, etc., and sheep, mice, horses, etc. are intermediate hosts. Mature Echinococcus lays eggs in the small intestine of the final host, and is excreted with feces, and is swallowed by the intermediate host. After eating, through the action of digestive juice, the larvae come out of their shells and become Hexacarpa. These animals complete the life cycle of Echinococcus after being swallowed by the final host. [0003] Cystic echinococcosis is mainly distributed in the liver, accounting for 75%-78%, and accounting for 8.5%-14.5% in the lungs. Others such as brain, spleen, kidney, bones and female ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62G01N33/569
Inventor 张雪飞王虎马俊英高琦刘佳郄霜蔡辉霞刘培运王威雷雯
Owner 青海省地方病预防控制所
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