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Novel hydatidosis diagnosing antigen and application thereof

A technology of Echinococcus granulosus and antigen, applied in the direction of application, anti-peptide structure protease inhibitor immunoglobulin, protease inhibitor, etc., can solve the problem of low sensitivity, reduced sensitivity, and inability to meet the requirements of actual diagnosis, etc. question

Active Publication Date: 2017-08-11
中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心 +1
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AI Technical Summary

Problems solved by technology

The natural antigen B reported so far has certain sensitivity and specificity for the diagnosis of cystic echinococcosis, but the sensitivity is often low, but peptide antigens prepared on the basis of AgB, such as P176, P175, P177, etc., can improve the specificity of diagnosis. resistance, but often with reduced sensitivity
[0012] In addition, new diagnostic antigens have been discovered one after another, but so far, the sensitivity and specificity of these reported antigens for the detection of echinococcosis are still very limited in actual diagnosis, and cannot be detected. Meet the requirements of practical diagnosis

Method used

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  • Novel hydatidosis diagnosing antigen and application thereof
  • Novel hydatidosis diagnosing antigen and application thereof
  • Novel hydatidosis diagnosing antigen and application thereof

Examples

Experimental program
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preparation example Construction

[0073] The preparation method of the antigen of the present invention has the following steps:

[0074] (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;

[0075] (2). Host cells cultured in a suitable medium;

[0076] (3). Isolate and purify the antigen of the present invention from culture medium or cells.

[0077] 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

[0097] Example 1: Cloning of Echinococcus granulosus cysteine ​​protease inhibitor (EgCystatin) gene

[0098] 1.1 Amplification of EgCystatin gene:

[0099] 1.1.1 Target gene primer design:

[0100] According to the nucleotide sequence of EgCystatin (GenBank accession number: LK028576), utilize PrimerPremier 5 software to design primers:

[0101] EgCystatin-F:5'-AATGGGTCGC GGATCC TGCAGACAGCAAGAGCG-3' (SEQ ID NO.: 3), the italicized sequence is homologous to the vector, and the underline is the XhoI restriction site.

[0102] EgCystatin-R:5'-GGTGGTGGTG CTCGAG TTATAGGGCTGTGGGGTCTT-3' (SEQ ID NO.: 4), italics are sequences homologous to the vector, and the underline is the BamHI enzyme cutting site (synthesized by Shanghai Huada Company).

[0103] 1.1.2PCR amplification of the target gene:

[0104] Using Echinococcus granulosus cDNA as a template, the target gene was amplified by PCR, and the PCR reaction system was as follows:

[0105] dNTP Mixture (2.5mM each)...

Embodiment 2

[0122] Embodiment 2: Construction of target gene recombination plasmid

[0123] 2.1 Preparation of empty plasmid pET-28a:

[0124] 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:

[0125] (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.

[0126] (2) Randomly pick 4 single colonies, pipette them into 2μl LB culture medium respectively, take 3μl as a template, and carry out PCR verification. The system is as follows:

[0127] Template DNA 3.0μl Primer F (10μM each) 1.0μl Primer R (10μM each) 1.0μl 2 x MasterMix 12μl wxya 2 o

8μl total capacity 25.0μl ...

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Abstract

The invention provides a novel hydatidosis diagnosing antigen and an application thereof. Specifically, the invention provides echinococcus granulosus cystatin abbreviated to EgCystatin or genes of echinococcus granulosus or an application of a detection reagent of echinococcus granulosus. The EgCystatin, the genes of echinococcus granulosus and the detection reagent are used for preparing a positive antigen of echinococcus granulosus and / or used for preparing a reagent or a kit for diagnosing echinococcus granulosus. The antigen has very high diagnosis sensitivity and specificity to echinococcus granulosus and has the diagnostic efficiency higher than 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 funded by the following funds: National Natural Science Foundation of China (81201315); National Major Science and Technology Project (2009ZX10004-302); Gansu Provincial Science and Technology Support Program (1304FKCA120); Key Laboratory of Parasitic Pathogens and Vector Biology of Health Planning Commission is open Topic (WSBKTKT201305). Background technique [0004] Echinococcus granulosus (Echinococcus granulosus) larvae (Echinococcus) parasitize the human body and cause Cyst echinococcosis (Cystichydatid disease), also known as cystic echinococcosis (Cystichydatid disease), is a serious hazard Human health and life safety, and zoonotic diseases that affect social and economic develo...

Claims

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Application Information

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IPC IPC(8): C07K14/81C12N15/15C07K16/38G01N33/68
CPCC07K14/8139C07K16/38G01N33/68
Inventor 张颋陈英胡薇冯宇陈军虎王东贾利芳莫筱瑾陈绅波徐斌
Owner 中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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