Fasciola hepatic fasciola hypothetical protein gene and colloidal gold immunochromatography test paper

A technology of Fasciola hepatica and hypothetical protein, applied in the field of immunology, can solve the problem of lack of immunodiagnosis methods for Fasciola hepatica, and achieve the effects of clearly visible results, high sensitivity and short time.

Active Publication Date: 2020-04-28
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventional detection method of fascioliasis is to check whether there are fasciola hepatica larvae in the feces to confirm the diagnosis. There are many inconveniences in practical application. At present, there is a lack of simple and rapid immunodiagnostic methods for fasciola hepatica

Method used

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  • Fasciola hepatic fasciola hypothetical protein gene and colloidal gold immunochromatography test paper
  • Fasciola hepatic fasciola hypothetical protein gene and colloidal gold immunochromatography test paper
  • Fasciola hepatic fasciola hypothetical protein gene and colloidal gold immunochromatography test paper

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Cloning and expression of the hypothetical protein D915_11650 recombinant protein gene of Fasciola hepatica

[0046] 1. Cloning of inserted gene from lambda phage

[0047] The sequence of primers for inserting gene fragment amplification was synthesized by Shanghai Shenggong, as follows:

[0048] F: 5'-CTCGGGAAGCGCGCCATTGTGTTGGT-3';

[0049] R: 5'-ATACGACTCACTATAGGGCGAATTGGCC-3';

[0050] 2. Open reading frame PCR amplification

[0051] According to the open reading frame gene sequence, use Primer5.0 software for primer design, and the restriction enzymes are SacI and Hind Ⅲ:

[0052] F: 5’-C GAGCTC ATGGTTCTTGCTGAGGAACGACAG-3’;

[0053] R: 5’-CCC AAGCTT TCAACTGACCATATCACCGAGATC-3’;

[0054] Open reading frame PCR amplification using the amplification product of Fasciola hepatica hypothetical protein D915_11650 gene as template

[0055] 3. Construction of cloning vector of Fasciola hepatica hypothetical protein D915_11650 gene

[0056] The target fragment is ligated with pMD-18T ve...

Embodiment 2

[0059] Example 2 Preparation of colloidal gold test strips for detection of Fasciola hepatica infection

[0060] 1. Method

[0061] (1) Sintering of colloidal gold

[0062] Using trisodium citrate reduction method, to 99mL ddH 2 Add 1mL 1% chloroauric acid to O, add 1mL 1% trisodium citrate after boiling to prepare 40nm colloidal gold particles;

[0063] (2) Determination of the optimal pH of gold-labeled antibody

[0064] Use K 2 CO 3 Gradient marking method, by observing the color change, determine the best PH of the gold label;

[0065] (3) Preparation of gold-labeled antibody

[0066] Use the best K 2 CO 3 The added amount is labeled with colloidal gold, and the labeled gold-labeled antibody is dissolved in the compound solution and stored at 4°C for later use or dropped directly on the gold-labeled pad;

[0067] (4) Determination of the coating concentration of T-line hypothetical protein D915_11650 on NC membrane

[0068] Dilute the hypothetical T-line protein D915_11650 protein to 2,...

Embodiment 3

[0083] Preparation of colloidal gold test strips

[0084] Nitrocellulose membrane 21mm, sample pad 24mm, gold label pad 9mm, absorbent paper 32mm are pasted on the PVC bottom in sequence from bottom to top, sample pad presses gold label pad 2mm, gold label pad presses NC film 2mm, absorbent paper presses NC film 2mm; After pasting them in sequence, use a paper cutter to cut the assembled test paper into test strips each with a width of 4mm. T-line coats the hypothetical gene D915_11650 protein of Fasciola hepatica and C-line coats rabbit anti-SPA antibody. .

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Abstract

The invention discloses a hypothetical protein gene of fasciola hepatica. Meanwhile, the invention further provides colloidal gold immunochromatography test paper prepared from the hypothetical protein gene of the fasciola hepatica. A fasciola hepatica infection detection colloidal gold test paper strip prepared by the hypothetical protein gene of the fasciola hepatica, the test paper has the characteristics of simplicity, convenience, easiness in operation, high sensitivity, high specificity, short time and the like and clear and visible result and is suitable for clinical field detection andlarge-scale epidemiological investigation.

Description

Technical field [0001] The invention discloses a hypothetical protein gene of Fasciola hepatica, and also provides a colloidal gold immunochromatographic test paper made of the hypothetical protein gene of Fasciola hepatica, which is used for serum detection of Fasciola hepatica infection in cattle and sheep, and belongs to the technical field of immunology . Background technique [0002] Fasciola hepatica parasitizes in the liver and bile ducts of ruminants such as cattle and sheep, causing acute or chronic hepatitis and cholangitis in the host. The disease has a worldwide distribution and is often endemic. Rainy years can promote the epidemic of the disease and seriously threaten the health of humans and animals and hinder the development of animal husbandry. The conventional method of detection of Fasciola hepatica is to check whether the feces contains Fasciola hepatica larvae to confirm the diagnosis. There are many inconveniences in practical application. At present, there...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/532
CPCG01N33/532
Inventor 李建华刘天张西臣宫鹏涛李新杨举李赫
Owner JILIN UNIV
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