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Trichina Ts27 gene, encoding protein and application thereof

A protein and amino acid technology, applied in genetic engineering, plant genetic improvement, peptide/protein components, etc., can solve problems such as poor specificity, inability to subculture Trichinella spiralis pathogens, and limited antigen acquisition

Inactive Publication Date: 2011-09-14
CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ES and parasite antigens have cross-reactions with Paragonimus, Clonorchis sinensis, Schistosoma japonicum and cysticercosis, so the specificity is poor
Although the recombinant protein can improve the specificity, its sensitivity is not high
[0004] Because the Trichinella spiralis pathogen cannot be subcultured in large quantities in vitro, the acquisition of antigens is limited; coupled with the complexity and diversity of Trichinella spiralis antigens, there is currently no good antigen with high sensitivity and high specificity as a candidate antigen for diagnosis

Method used

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  • Trichina Ts27 gene, encoding protein and application thereof
  • Trichina Ts27 gene, encoding protein and application thereof
  • Trichina Ts27 gene, encoding protein and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1 Cloning and sequence analysis of the Ts27 gene of Trichinella spiralis

[0069] 1. Preparation of Screening Serum

[0070] Collect Trichinella muscle larvae (international standard strain ISS533), experimental pigs (Wuzhishan miniature pig), 20kg / head, a total of 4 animals, orally infect 20,000 Trichinella muscle larvae, New Zealand white rabbits and BALB / C mice Two of them were orally infected with 4000 and 500 Trichinella muscle larvae respectively. After 4 weeks, the serum of artificially infected pigs, infected rabbits and infected mice were obtained. The serum titers were determined by ELISA, and the serum titers of infected pigs were more than 1:10000, and the serum sera of infected rabbits and infected mice were more than 1:1000.

[0071] 2. Immunoscreening of Expression Libraries

[0072] Trichinella adult λZAPII cDNA expression library was purchased from Liu Mingyuan (reference "Construction and Screening of Adult and Neonatal Larval Gene Library of...

Embodiment 2

[0078] Example 2 Preparation of Ts27 Gene Recombinant Protein and Immune Serum

[0079] 1. Prokaryotic expression and purification of Ts27 gene

[0080] (1) Amplify the Ts27 gene:

[0081] The positive clone pBluescript / Ts27 bacterium liquid that obtains with embodiment 1 is carried out whole bacterium PCR amplification as template, and primer is

[0082] 5'-CGGGATCCGAAAA TATGGCACATAGCAATG-3' (SEQ ID NO: 3); and 5'-CGCTCGAGTGCACGGTTA TTGAAAACAGC-3' (SEQ ID NO: 4).

[0083] Amplification conditions: 95°C for 5min; 94°C for 1min, 55°C for 1min, 72°C for 2min, 35 cycles; 72°C for 10min.

[0084] (2) The amplified and purified Ts27 gene fragment was digested with BamH I and Xho I, and then subcloned into the prokaryotic expression vector PET-28a(+). Transform Escherichia coli BL21 strain with the correct recombinant plasmid. After being induced by 1.0mM IPTG at 150rpm at 37°C for 3 hours, divide the bacterial solution into 50ml centrifuge tubes, centrifuge at 11000rpm, 4°C for ...

Embodiment 3

[0087] The immune characteristic of embodiment 3Ts27 gene recombinant protein

[0088] 1. Enzyme-linked immunosorbent assay (ELISA) detection

[0089] Use 1 μg of the Ts27 gene recombinant protein prepared in Example 2 as an antigen-coated 96-well microtiter plate, the primary antibody is the serum to be tested, and the serum to be tested is the serum of Trichinella patients (35 cases, provided by Huazhong University of Science and Technology, working concentration 1:200), rabbit and mouse serum (2 examples each, prepared in Example 1, working concentration is 1:1000) infected with Trichinella spiralis, pig serum infected with Trichinella spiralis (2 examples, prepared in Example 1, working concentration Both are 1:10000). The secondary antibody was the corresponding secondary antibody labeled with horseradish peroxidase (goat anti-mouse, goat anti-rabbit, goat anti-human secondary antibody, purchased from Beijing Zhongshan Jinqiao Biological Company, rabbit anti-pig secondar...

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Abstract

The invention relates to a trichina gene Ts27 and a protein encoded by the same and also relates to a recombinant vector and a recombinant cell containing the gene Ts27 and an antibody specifically combined with the protein encoded by the gene Ts27. The invention also relates to an application of the gene Ts27, the protein encoded by the gene Ts27 and the antibody to preparation of a detection reagent for diagnosing trichinization of humans and animals, and a detection kit. After the gene Ts27 recombinant protein is immune to animals, an antibody with a high titer can be generated; and the gene Ts27 recombinant protein can be specifically identified by animal or human serums infected by trichinas.

Description

technical field [0001] The present invention relates to a Trichinella gene, in particular to a Trichinella Ts27 gene, a protein encoded by the gene, and uses of the gene and protein. Background technique [0002] Trichinella spiralis (Trichinella spiralis for short) can infect humans and more than 150 kinds of animals. Trichinellasis caused by it is a zoonotic parasitic disease with a global distribution, which seriously threatens human health and Huge economic losses to animal husbandry. The life history of Trichinella spiralis is briefly described as follows: When the human or animal host eats raw or semi-raw meat containing Trichinella capsules (pork, dog meat, etc.), the larvae in the capsules escape under the action of gastric juice and intestinal juice and develop into adults in the small intestine. After mating, the females produce new larvae. The newborn larvae invade the intestinal mucosal lymphatic vessels or veins, and then reach the host striated muscle with th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N15/11C07K14/435C07K16/18G01N33/577G01N33/53A61K38/17A61K39/395A61P33/10
Inventor 诸欣平杨静杨雅平秦佳佳
Owner CAPITAL UNIVERSITY OF MEDICAL SCIENCES
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