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Recombinant antigenic protein for diagnosing echinococcosis granulosus, preparation method thereof and use thereof

A technology for echinococcosis and recombinant antigenic protein, which is applied in the field of preparation of the above-mentioned recombinant antigenic protein, recombinant protein, and recombinant antigenic protein for the diagnosis of echinococcosis, which can solve the unsatisfactory treatment effect of echinococcosis, etc. question

Active Publication Date: 2012-08-29
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 granulosus in my country is more than 50 million people, but the current treatment effect on echinococcosis is not satisfactory, so early diagnosis is crucial for the timely diagnosis of human cystic echinococcosis (CE). Treatment is crucial, and specific immunological diagnosis plays an important role in the diagnosis of CE

Method used

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  • Recombinant antigenic protein for diagnosing echinococcosis granulosus, preparation method thereof and use thereof
  • Recombinant antigenic protein for diagnosing echinococcosis granulosus, preparation method thereof and use thereof
  • Recombinant antigenic protein for diagnosing echinococcosis granulosus, preparation method thereof and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of Echinococcus granulosus Recombinant Antigen Protein

[0032] 1 material

[0033] 1.1 Echinococcus granulosus

[0034] Echinococcus granulosus cysts in sheep liver were collected from the slaughterhouse in Qinghai Province, and the protoscole of Echinococcus granulosus from Qinghai (sheep source) was isolated and collected under aseptic conditions.

[0035] 1.2 Serum samples to be tested

[0036] Qinghai Provincial Institute of Endemic Disease Prevention and Control provided 60 sera from confirmed CE patients, 37 sera from alveolar echinococcosis (AE) patients, 11 sera from cysticercosis patients, and the Institute of Parasitic Diseases Prevention and Control of the Chinese Center for Disease Control and Prevention. Provide 33 serums of healthy people, 5 serums of cysticercosis patients, 7 serums of liver fluke patients, 4 serums of schistosomiasis patients, and 4 serums of toxoplasmosis patients.

[0037] 1.3 Main reagents and tool enzymes

...

Embodiment 2

[0058] Example 2 Western-blot identification of PET28a-EgEPC1 recombinant protein

[0059] 1. Western-blot identification method

[0060] After the purified PET28a-EgEPC1 recombinant protein (produced by Example 1) was subjected to 15% SDS-PAGE electrophoresis, it was transferred to a nitrocellulose membrane; the membrane was cut into strips and placed in 5% skim milk powder solution to block overnight, Wash with PBS-T 3 times, 5 min each time; react with the mixed serum of normal healthy people, the mixed serum of CE patients, and the mixed serum of AE patients (1:100 dilution), shake gently at room temperature for 2 h, wash 3 times with PBS-T, each time 5min; add goat anti-human HRP-IgG (1:1000) and shake gently at room temperature for 2h, wash with PBS-T 3 times, 5min each time; add freshly prepared DAB combined chromogenic solution until the target band appears, stop with deionized water reaction.

[0061] 2. Western-blot identification and analysis results of recombinan...

Embodiment 3

[0063] Example 3 Diagnosis Effect Experiment of Echinococcus granulosus Recombinant Antigen Protein

[0064] 1. ELISA serological evaluation of recombinant antigens

[0065] Multiple parts of serum CE and healthy human serum 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 0.5 μg / ml by square matrix titration, and the working concentration of goat anti-human IgG horseradish peroxidase marker was 1:14000. O-phenylenediamine substrate (OPD) color development, A 490 . The mean absorbance of 33 cases of healthy human serum was 2 times SD as the positive judgment value.

[0066] 2. Statistical analysis of data

[0067] The experimental data were collated and statistically analyzed using Excel spreadsheet processing software.

[0068] 3. Analysis and evaluation of serological test results of recombinant antigens

[0069]...

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Abstract

The invention discloses a recombinant antigenic protein for diagnosing echinococcosis granulosus (having an amino acid sequence represented by SEQIDNo.1). In addition, the invention also discloses the preparation method of the recombinant antigenic protein, which comprises: amplifying an EgEPC1 gene by using RT-PCR; cloning the EgEPC1 gene in a pGEM-T vector; connecting the EgEPC1 gene with an expression vector PET28a(+) to form a recombinant plasmid PET28a-EgEPC1; transforming the recombinant plasmid PET28a-EgEPC1 to Escherichia coli BL21(DE3) and expressing the recombinant protein through IPTG induction; and identifying a purified recombinant antigen by using SDS-PAGE and Western blotting. In addition, the invention also discloses the diagnosis use of the recombinant antigenic protein. Experiments show that the recombinant antigenic protein of the invention has the advantages of high sensibility and specificity for the diagnosis of echinococcosis granulosus, and has a promising application prospect in the diagnosis of the echinococcosis granulosus.

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. The prevalence of echinococcosis in my country is serious, accounting for about 44% of the total area of ​​the country. It is estimated that the number of echinococcosis patients in the country is about 380,000, especially in the western region. At present, the diagnosis of CE is mainly through physical imagi...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/11C12N15/12C12N15/70C12Q1/68G01N33/53
Inventor 曹建平蔡辉霞沈玉娟韩秀敏胡媛王虎卢潍媛徐馀信官亚宜
Owner STATION OF VIRUS PREVENTION & CONTROL CHINA DISEASES PREVENTION & CONTROL CENT
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