A Schistosoma japonicum immune epitope recombinant protein and its application

A technology of recombinant protein and schistosomiasis, applied in the field of bioengineering, can solve the problems of lower detection cost, limited specificity and sensitivity of schistosomiasis infection detection method, and achieve good sensitivity effect

Active Publication Date: 2022-03-25
SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is to overcome the limited specificity and sensitivity of the current Schistosoma japonicum infection detection method, and / or reduce the detection cost

Method used

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  • A Schistosoma japonicum immune epitope recombinant protein and its application
  • A Schistosoma japonicum immune epitope recombinant protein and its application
  • A Schistosoma japonicum immune epitope recombinant protein and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Bioinformatics analysis and epitope prediction of schistosome-specific extracellular vesicle proteins

[0038] Through mass spectrometry analysis and data query of the proteins of Schistosoma japonicum extracellular vesicles, 7 protein molecules specific to Schistosoma japonicum and with diagnostic potential were finally obtained. DNASTAR software was used to predict possible epitopes, and selected proteins with good hydrophilicity and Peptides with strong antigenicity: epitope peptide 68 (shown in SEQ ID NO.1) and epitope peptide 71 (shown in SEQ ID NO.2), chemically synthesized by Gill Biochemical (Shanghai) Co., Ltd.

Embodiment 2

[0039] Cloning, expression and purification of embodiment 2 recombinant tandem epitope peptide

[0040] The nucleic acid sequence cDNA fragment encoding the tandem epitope peptide (sequence shown in SEQ ID NO.4) was synthesized by Shanghai Jieli Biotechnology Co., Ltd., and then it and the expression vector pET32a (+) were all treated with restriction endonucleases KpnI and HindIII double enzyme digestion, the digested products were gel recovered and ligated, and then the recombinant plasmid containing the correct insertion was transformed into Escherichia coli BL21 competent cells. The screened positive recombinant bacteria were inoculated in LB ampicillin medium, when OD 600nmWhen the value is 0.6-0.8, add 1.0mMIPTG and induce expression at 37°C for 8 hours. Then, the enriched recombinant protein was purified by His·Bind Resin purification kit, and separated and identified by SDS-PAGE on 12.0% protein gel. The SDS-PAGE analysis results of the induced recombinant protein an...

Embodiment 3

[0041] Example 3 Preparation of mice and rabbit serum samples infected with Schistosoma japonicum for different days and preparation of soluble egg antigen (SEA)

[0042] Clean-grade mice and New Zealand white rabbits were infected with about 200 and 2000 cercariae of Schistosoma japonicum through abdominal skin patch, respectively. At intervals of 7 days after infection, blood was collected from the orbit of the mouse and from the vein of the ear margin of the rabbit. After collecting whole blood, let it stand at room temperature for 30 minutes, then centrifuge at 1,000-2,000×g for 20 minutes at 4°C to collect serum.

[0043] Rabbit livers infected with Schistosoma japonicum cercariae for 42 days were collected, cut into small pieces, put into a high-speed tissue masher, added an appropriate amount of phosphate buffered saline (PBS) pre-cooled at 4°C, and homogenized at high speed. Pass the homogenate through 60, 80, 120, 160 and 200 mesh screens in turn, collect the filtrat...

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Abstract

The invention discloses a Schistosoma japonicum immune epitope recombinant protein, which is represented by epitope peptide 68 shown in SEQ ID NO.1, epitope peptide 71 shown in SEQ ID NO.2 or SEQ ID NO.3. The tandem epitope peptides shown. The invention also discloses the application of the above-mentioned immune epitope recombinant protein of Schistosoma japonicum in the preparation of products for diagnosing Schistosoma japonicum. The Schistosoma japonicum immune epitope recombinant protein of the invention can effectively and rapidly detect the infection of the Schistosoma japonicum, and has good sensitivity in detecting the early infection of the Schistosoma japonicum.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a Schistosoma japonicum immune epitope recombinant protein and its application. Background technique [0002] Schistosomiasis is a zoonotic parasitic disease caused by schistosome infection, which poses a serious threat to human and animal health. Timely and accurate diagnosis is crucial for disease control. The conventional method is to detect the presence of eggs in the feces, but this method is not sensitive and prone to false negative results. Although PCR-based methods have achieved more accurate results, it is still difficult to achieve large-scale applications due to issues such as cost and experimental conditions. The ELISA-based antigen-antibody detection method has less cross-contamination, and is reasonably priced and easy to operate, and has become one of the better methods for the diagnosis of schistosomiasis. [0003] Extracellular vesicles are tiny vesicle...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/70G01N33/68
CPCC07K14/43559C12N15/70G01N33/6893G01N2333/43547G01N2800/42
Inventor 程国锋陈永军夏天奇李雪
Owner SHANGHAI VETERINARY RES INST CHINESE ACAD OF AGRI SCI
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