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Novel positive antigen of echinococcus granulosus and application of antigen

A technology of Echinococcus granulosus and Echinococcus granulosus, applied in the field of new antigen diagnosis of Echinococcus granulosus

Pending Publication Date: 2015-05-27
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Echinococcus granulosus LDH as a new effective antigen for the diagnosis of echinococcosis has not been reported yet

Method used

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  • Novel positive antigen of echinococcus granulosus and application of antigen
  • Novel positive antigen of echinococcus granulosus and application of antigen
  • Novel positive antigen of echinococcus granulosus and application of antigen

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preparation example Construction

[0070] The preparation method of the antigen of the present invention has the following steps:

[0071] (1). Transform or transduce a suitable host cell with a polynucleotide (or variant) encoding the positive antigen of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0072] (2). Host cells cultured in a suitable medium;

[0073] (3). Isolate and purify the antigen of the present invention from culture medium or cells.

[0074] In the present invention, polynucleotide sequences can be inserted into recombinant expression vectors. Methods well known to those skilled in the art can be used to construct expression vectors containing the antigen-encoding DNA sequence and appropriate transcriptional / translational control signals. A vector comprising the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express the protein.

[007...

Embodiment 1

[0094] Example 1: Cloning of Echinococcus granulosus lactate dehydrogenase (lactate dehydrogenase, EgLDH) gene

[0095] 1.1 Amplification of EgLDH gene:

[0096] 1.1.1 Target gene primer design:

[0097] According to the known EgLDH coding full-length sequence (GenBank accession number: HM748917.1), use Primer Premier 5 software to design primers:

[0098] EgLDH-F:5'-AATGGGTCGC GGATCC ATGTCTGTGGAGGGGTTG-3' (SEQ ID NO.: 3), italics are homologous sequences with the vector, and the underline is the XhoI restriction site.

[0099] EgLDH-R:5'-GGTGGTGGTG CTCGAG TTATCACCACTTGATGCCTGC-3' (SEQ ID NO.: 4), italics are sequences homologous to the vector, and the underline is the BamHI restriction site (synthesized by BGI).

[0100] 1.1.2PCR amplification of the target gene:

[0101] Using Echinococcus granulosus cDNA as a template, the target gene was amplified by PCR, and the PCR reaction system was as follows:

[0102]

[0103] The reaction conditions are as follows:

[01...

Embodiment 2

[0119] Example 2: Linearization of vectors

[0120] 2.1 Preparation of empty plasmid pET-28a:

[0121] Plate the carrier bacteria (Novagen) of the pET-28a plasmid, extract the plasmid after identifying a single colony by PCR, and linearize the plasmid by double enzyme digestion. The operation is as follows:

[0122] (1) Dilute 1 μl of the seed-preserving carrier bacterial solution with 500 μl of ddH 2 O diluted, pipetted 100 μl plated onto Kana-resistant LB solid medium. Place at 37°C for about 1h until the surface liquid is absorbed, invert the plate, and incubate at 37°C for 16h.

[0123] (2) Randomly pick 4 single colonies, pipet them into 20 μl LB culture medium respectively, take 3 μl as a template, and carry out PCR verification. The system is as follows:

[0124]

[0125] Reaction conditions:

[0126]

[0127] After the reaction, the PCR product was subjected to 1% agarose gel electrophoresis (2 μl EB substitute / 40ml), 130V, 25min.

[0128] (3) Add the bacter...

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Abstract

The invention provides a novel positive antigen of echinococcus granulosus and an application of the antigen and in particular related to an echinococcus granulosus lactated dehydrogenase (EgLDH), a gene or an application of a detection reagent of the echinococcus granulosus lactated dehydrogenase. The echinococcus granulosus lactated dehydrogenase or the gene thereof is applied to the aspect as follows: (i) preparation of positive antigen of echinococcus granulosus or echinococcosis granulosis cyst; and / or (ii) preparation of a reagent or a kit for diagnosing echinococcus granulosus or echinococcosis granulosis cyst. The antigen is highly excellent in diagnostic sensitivity and specificity on the echinococcus granulosus; the diagnostic efficiency of the antigen is highly superior to that of other conventional echinococcus granulosus antigens.

Description

technical field [0001] The invention relates to the field of diagnosis of parasitic diseases. Specifically, the present invention relates to a novel antigen useful for the diagnosis of Echinococcus granulosus (or Echinococcus granulosus). [0002] related funding [0003] This invention is supported by the following funds: National Natural Science Foundation of China (81201315); China Postdoctoral Fund (2014M551331); National Major Science and Technology Project (2009ZX10004-302); Youth Fund of China Center for Disease Control and Prevention (2013A103); Shanghai Municipal Health and Family Planning Committee scientific research project (20124174). Background technique [0004] Echinococcus granulosus (Echinococcus granulosus) larvae (Echinococcus) parasitize the human body and cause Cyst echinococcus, also known as Cystic hydatid di sease, is a Zoonotic diseases that seriously endanger human health and life safety and affect social and economic development are the main dis...

Claims

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Application Information

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IPC IPC(8): C12N9/04C12N15/53C07K16/40C07K19/00G01N33/53C12Q1/68
CPCC12N9/0006G01N33/573
Inventor 张颋胡薇陈英贾利芳冯正陈军虎刘杰奥·乌力吉徐斌莫筱瑾
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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