Recombinant antigen protein for diagnosing echinococcosis granulosa as well as preparation method and application thereof

A technology for echinococcosis granulosus and recombinant antigenic protein, which is applied to the preparation of the above-mentioned recombinant antigenic protein, the recombinant antigenic protein for diagnosing echinococcosis granulosus, and the field of recombinant proteins, and can solve the therapeutic effect of echinococcosis granulosus. Not ideal, etc.

Inactive Publication Date: 2013-01-09
STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] According to incomplete statistics, the population threatened by echinococcosis in my country is more than 50 million people, but the current treatment effect on echinococcosis (cystic echinococcosis) is not ideal, so early diagnosis is very important for people. Timely treatment of cystic echinococcosis (CE) is crucial, and specific immunological diagnosis plays an important role in the diagnosis of CE

Method used

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  • Recombinant antigen protein for diagnosing echinococcosis granulosa as well as preparation method and application thereof
  • Recombinant antigen protein for diagnosing echinococcosis granulosa as well as preparation method and application thereof
  • Recombinant antigen protein for diagnosing echinococcosis granulosa as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Preparation of Echinococcus granulosus Recombinant Antigen Protein

[0033] 1 material

[0034] 1.1 Echinococcus granulosus

[0035] Echinococcus granulosus cysts from sheep liver were collected from slaughterhouses in Qinghai Province, and the protoscole of Echinococcus granulosus from Qinghai (sheep source) was isolated and collected under aseptic conditions.

[0036] 1.2 Serum samples to be tested

[0037]32 serums from patients with cystic echinococcosis (CE), 24 serums from patients with alveolar echinococcosis (AE), 5 serums from patients with Schistosoma japonicum, and serum from patients with Toxoplasma gondii provided by the Institute of Parasitic Disease Prevention and Control, Chinese Center for Disease Control and Prevention 5 samples, 5 serum samples from Clonorchis sinensis patients, 4 serum samples from cysticercosis patients, 2 serum samples from Paragonimia sinensis patients and 16 healthy human serum samples.

[0038] 1.3 Main reagents and...

Embodiment 2

[0059] Western-blot identification of embodiment 2PET28a-EgENO recombinant protein

[0060] 1. Western-blot identification method

[0061] The purified PET28a-EgENO recombinant protein (prepared in Example 1) was electrophoresed by 10% SDS-PAGE and transferred to PVDF membrane (polyvinylidene fluoride membrane, polyvinylidene fluoride); the membrane was placed in 5% degreased The milk powder solution was blocked for 1 hour, mixed with cystic echinococcosis patient serum (diluted 1:200) to react, overnight at 4°C, TBST (TBST contains Tris-Hcl, NaCl, and tween20, which are commonly used in Western-blot a buffer solution) for 3 times, 5 min each time; add donkey anti-human HRP-IgG (1:10000) and shake gently at room temperature for 1 h, wash 3 times with TBST, 5 min each time; add freshly prepared DAB (diaminobenzidine ) combined with the chromogenic solution until the target band appeared, and deionized water was used to terminate the reaction.

[0062] 2. Western-blot identifi...

Embodiment 3

[0064] Example 3 Diagnosis Effect Experiment of Echinococcus granulosus Recombinant Antigen Protein

[0065] 1. ELISA serological evaluation of recombinant antigens

[0066] A number of cystic echinococcosis patient sera and healthy human sera were mixed in equal amounts to prepare positive and negative serum controls. The serum was diluted 1:100, and the working concentration of the recombinant antigen (prepared in Example 1) was determined to be 10 μg / ml by square array titration, and the working concentration of donkey anti-human HRP-IgG was 1:10000. DAB color, test A 450 . The 2 times SD of the mean absorbance of 16 cases of healthy human serum was used as the positive judgment value.

[0067] 2. Statistical analysis of data

[0068] The experimental data were collated and statistically analyzed using Excel spreadsheet processing software.

[0069] 3. Analysis and evaluation of serological test results of recombinant antigens

[0070] Using the recombinant antigen Eg...

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Abstract

The invention discloses a recombinant antigen protein (of which the amino acid sequence is shown as SEQ ID NO:1) for diagnosing echinococcosis granulose. Moreover, the invention further discloses a preparation method of the recombinant antigen protein. The method comprises the following steps of: amplifying an EgENO gene by adopting RT-PCR (Reverse Transcription-Polymerase Chain Reaction); cloning the EgENO gene into an expression vector PET28a (+) for constructing a recombined plasmid PET28a-EgENO; converting into escherichia coli BL21(DE3); inducing the expression of a recombinant protein through IPTG (Isopropyl-beta-d-Thiogalactoside); and identifying a purified recombinant antigen by using SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) and Western blotting. In addition, the invention further discloses a diagnosis application of the recombinant antigen protein. As proved by an experiment, the recombinant antigen protein has the advantages of high sensitivity, high specificity and the like for the diagnosis of the echinococcosis granulosa, and has a wide application prospect on the aspect of diagnosis of the echinococcosis granulosa.

Description

technical field [0001] The invention belongs to the field of biological detection, and specifically relates to a recombinant protein, in particular to a recombinant antigen protein for diagnosing echinococcosis granulosus; in addition, the invention also relates to the preparation method and application of the above recombinant antigen protein. Background technique [0002] Echinococcus granulosus, also known as cystic echinococcosis (CE), is a zoonotic parasitic disease caused by the larvae of Echinococcus granulosus, which seriously endangers human health and the development of animal husbandry. It has become an important public health and economic problem worldwide. my country is one of the countries with the highest incidence of cystic echinococcosis, among which Xinjiang, Qinghai, Gansu, Ningxia, northern Xichuan and other places in the west are the most serious. The epidemic area accounts for more than 44% of the total area of ​​the country, seriously affecting the agr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/11C12N15/12C12N15/70C12Q1/68G01N33/68
Inventor 曹建平王莹沈玉娟袁忠英周何军徐馀信张璟王燕娟伍卫平
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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