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Echinococcosis antigen gene (egG1Y162 antigen gene), and recombinant protein and use thereof

An egg1y162, antigen gene technology, applied in application, gene therapy, genetic engineering and other directions, can solve problems such as not achieving ideal results

Inactive Publication Date: 2009-07-08
XINJIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some progress has been made in the development of recombinant vaccines, the desired effect has not yet been achieved.

Method used

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  • Echinococcosis antigen gene (egG1Y162 antigen gene), and recombinant protein and use thereof
  • Echinococcosis antigen gene (egG1Y162 antigen gene), and recombinant protein and use thereof
  • Echinococcosis antigen gene (egG1Y162 antigen gene), and recombinant protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1: utilize polymerase chain reaction (PCR) technology to amplify egG1Y162 antigen gene of the present invention

[0020] First RNA (ribonucleic acid) extraction and PCR amplification of the egG1Y162 gene of the present invention: get 100 mg of protocercaria (derived from the liver of sheep infected with Echinococcus granulosus), add 1 milliliter of Trizol (can destroy the cell wall to release the ribonucleic acid) A kind of lysate that came out) was fully homogenized, and stood at room temperature for 5 minutes; added 0.2 ml of chloroform, oscillated for 15 seconds, and stood for 2 minutes; centrifuged at 4 degrees, 12000 g × 15 minutes, took the supernatant; Alcohol, mix the liquid in the tube gently, let it stand at room temperature for 10 minutes; centrifuge at 4 degrees, 12000 g × 10 minutes, discard the supernatant; add 1 ml of 75% ethanol, gently wash the precipitate, 4 degrees, 7500 g × 5 Minutes, discard the supernatant, dry it, and add 30 microliters...

Embodiment 2

[0024] Example 2: Obtaining a large number of egG1Y162 gene sequences of the present invention

[0025] The egG1Y162 gene of the present invention and the PUCm-T vector (a general cloning vector) were ligated overnight at 16°C under the action of T4 DNA ligase. The egG1Y162 target gene of the present invention is constructed on the PUCm-T vector, the PUCm-T / egY162 recombinant plasmid is constructed, and the recombinant plasmid is transformed into E. Coli DH5α (for cloning E. coli), and then plated and cultured overnight at 37 degrees , so that a large number of recombinant plasmids can be replicated in bacteria. 5 colonies were randomly selected, plasmids were extracted, and gene sequencing was carried out. The results were compared with online gene databases and found that the egG1Y162 gene of the present invention is a new gene found in Echinococcus granulosus.

Embodiment 3

[0026] Embodiment 3: obtain egG1Y162 recombinant protein of the present invention

[0027] It is necessary to construct the PET-41a / egG1Y162 prokaryotic expression plasmid, use EcoR I / Hind III to cut the PUCm-T / egG1Y162 recombinant plasmid and the prokaryotic expression plasmid pET41a, recover, connect, and transform into E.coli BL21 (a gene with expression function) coli) at 37°C overnight. The recombinant plasmid was amplified by PCR, identified by EcoRI / Hind III double enzyme digestion, and then sequenced. Sequencing correct PET-41a / egG1Y162 prokaryotic expression plasmid, 30 degrees, induced by IPTG (final concentration of 0.5 mmol / L isopropyl-β-D-thiogalactoside, protein inducer) for 0 hours, 2 Hours, 4 hours, 6 hours, collect the bacteria, centrifuge at 4 degrees, 12000 rpm for 10 minutes, and use 12% SDS-PAGE (polypropylene gel electrophoresis analysis) to detect, the mobility of proteins with different molecular weights is different, Then stain with Coomassie Brillia...

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Abstract

The present invention relates to the field of antigen gene technology, specifically to an echinococcosis antigen gene, namely egG1Y162 antigen gene and its recombinant protein and applications, wherein, the gene has a nucleotide sequence with a sequence 1. The invention obtains the echinococcosis antigen gene, namely egG1Y162 antigen gene and its recombinant protein from the granule echinococci, performs cloning, transforming and induce expression to the egg1y162 gene, the animal experiment further indicates that the egg1y162 recombinant protein vaccine has a protective effect on the infection of secondary granule echinococci to mice, becomes a candidate gene vaccine for preventing and controlling echinococcosis, and provides a new way for the development of practical vaccine.

Description

1. Technical field [0001] The invention relates to the technical field of antigen genes, and relates to an echinococcosis antigen gene, namely egG1Y162 antigen gene, its recombinant protein and application thereof. 2. Background technology [0002] Echinococcosis (cystic echinococcosis; CE), also known as echinococcosis, is a disease caused by the larvae of Echinococcus granulosus (Eg) parasitizing the human body and the liver, lungs and other organs of some animals. Severe zoonotic chronic parasitic disease. The measures to prevent echinococcosis are to control the source of infection and cut off the route of transmission. Although some progress has been made in the development of recombinant vaccines, ideal results have not yet been achieved. [0003] Japanese scholar Katoh [1, 2] found that some vaccine candidate proteins have secretion function and membrane surface anchoring function when studying Echinococcus multilocularis tapeworm. transmembrane region and is invol...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/11C07K14/435C12Q1/68G01N33/53A61K48/00A61K39/00A61P33/14
Inventor 丁剑冰曹春宝马秀敏吾拉木﹒马木提朱明林仁勇温浩
Owner XINJIANG MEDICAL UNIV
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