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

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

Active Publication Date: 2011-01-19
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 granulosa recombinant antigenic protein

[0032] 1 material

[0033] 1.1 Echinococcus granulosus

[0034] Echinococcus sacs from sheep liver were collected from slaughterhouses in Qinghai Province, and the protocephalus of Echinococcus granulosus was isolated and collected under sterile conditions.

[0035] 1.2 Serum samples to be tested

[0036] Qinghai Provincial Institute for Endemic Disease Control and Prevention provided 60 serum samples from patients with confirmed CE, 37 samples from patients with alveolar echinococcosis (AE), and 11 samples from patients with cysticercosis. Provided 33 sera from healthy people, 5 sera from cysticercosis, 7 sera from liver fluke disease, 4 sera from schistosomiasis, and 4 sera from toxoplasmosis.

[0037] 1.3 Main reagents and tool enzymes

[0038] Total RNA Isolation kit was purchased from Macherey-Nagel Company, Germany; AMV reverse transcriptase, TaqDNA polymerase, restriction endonucl...

Embodiment 2

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

[0059] 1. Western-blot identification method

[0060] The purified PET28a-EgEPC1 recombinant protein (made in Example 1) was electrophoresed on nitrocellulose membrane after electrophoresis by 15% SDS-PAGE; the membrane was cut into strips and placed in a 5% nonfat 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, CE patient mixed serum, and AE patient mixed serum (1:100 dilution), shake gently for 2 h at room temperature, and wash 3 times with PBS-T, each time 5min; add goat anti-human HRP-IgG (1:1000), shake gently at room temperature for 2h, wash 3 times with PBS-T, 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 recombinant proteins

[0062] The analy...

Embodiment 3

[0063] Example 3 Diagnostic effect test of Echinococcus granulosus recombinant antigen protein

[0064] 1. ELISA serological evaluation of recombinant antigens

[0065] Positive and negative serum controls were prepared by mixing equal amounts of serum CE and healthy human serum. Serum was diluted 1:100, and the working concentration of recombinant antigen (made in Example 1) was determined by square array titration to be 0.5 μg / ml, and the working concentration of goat anti-human IgG horseradish peroxidase marker was 1:14000. O-phenylenediamine substrate (OPD) color, test A 490 . The average absorbance value of 33 healthy people's serum was 2 times SD as the positive judgment value.

[0066] 2. Statistical analysis of data

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

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

[0069] Using recombinant antigen EPC1 to de...

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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, in particular to a recombinant protein, in particular to a recombinant antigen protein for diagnosing Echinococcus granulosus; in addition, the invention also relates to a 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 that seriously endangers human health and the development of animal husbandry. It has become an important public health and economic problem worldwide. Echinococcosis is seriously prevalent in my country, accounting for about 44% of the country's total area. It is estimated that the number of hydatid patients in the country is about 380,000, especially in the western region. At present, CE is mainly diagnosed by physical imaging, such as X-ray, B-ultrasound, tomography and n...

Claims

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Application Information

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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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