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A recombinant fusion protein of Schistosoma aegypti shsap and its application in the immunodiagnosis of schistosomiasis

A technology of fusion protein and schistosomiasis, applied in disease diagnosis, recombinant DNA technology, fusion polypeptide, etc., can solve problems such as differences and achieve good repeatability, high sensitivity, and good specificity

Active Publication Date: 2021-09-03
INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the large difference in sequence between the Schistosoma japonicum protein and the homologous protein of Schistosoma haematobium, the existing immunodiagnostic methods for Schistosoma japonicum cannot fully meet the needs of immunodiagnosis of Schistosoma haematobium

Method used

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  • A recombinant fusion protein of Schistosoma aegypti shsap and its application in the immunodiagnosis of schistosomiasis
  • A recombinant fusion protein of Schistosoma aegypti shsap and its application in the immunodiagnosis of schistosomiasis
  • A recombinant fusion protein of Schistosoma aegypti shsap and its application in the immunodiagnosis of schistosomiasis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Schistosoma haematobium ShSAP fusion protein gene synthesis and prokaryotic expression vector construction

[0028] The ShSAP gene encoded by the whole genome of Schistosoma haematobium was predicted using bioinformatics methods. The amino acid sequence of the Schistosoma haematobium ShSAP fusion protein is shown in the sequence table SEQ ID NO.1, and the ShSAP fusion protein was activated by the sphingolipid of Schistosoma haematobium haematobium from which the signal peptide sequence was removed. Protein-like protein 1 (ShSAP1) and Schistosoma haematobium saposin-like protein 2 (ShSAP2), the amino acid sequences of ShSAP1 protein and ShSAP2 protein are as shown in the sequence table SEQ ID NO.2 and SEQ ID NO.3. According to the codon preference of Escherichia coli, the ShSAP fusion protein coding gene was designed, and then the gene synthesis technology was used to prepare the ShSAP fusion gene of Schistosoma haematobium, whose nucleotide sequence was as show...

Embodiment 2

[0030] Example 2 Prokaryotic expression and purification of Schistosoma haematobium ShSAP fusion protein

[0031] Transform the pET28a-ShSAP recombinant plasmid with correct sequencing into expression competent cells Transetta (DE3), spread the transformed competent cells on LB medium plates (containing 50 μg / ml kanamycin), and culture overnight at 37°C; Positive clones were inoculated into 15 mL of LB liquid medium (containing 50 μg / ml kanamycin), cultured overnight at 37 ° C, and transferred 10 mL of medium into 1L LB medium (containing 50 μg / ml kanamycin) the next day, Continue to culture to OD 600nm When the value is 0.8, IPTG with a final concentration of 1 mM was added to induce expression for 4 hours, the cells were collected by centrifugation, and frozen at -80°C for future use.

[0032] Take a small amount of pre-induction and post-induction bacteria resuspended in PBS buffer, add SDS-PAGE sample buffer, mix well, boil in boiling water bath for 5min to denature the p...

Embodiment 3

[0035] Example 3 Antigenicity Detection of Schistosoma haematobium ShSAP Recombinant Protein

[0036] Take 200ng of ShSAP recombinant protein as a sample, and perform SDS-PAGE gel electrophoresis; transfer the protein in the PAGE gel to PVDF membrane by wet transfer method; seal the PVDF membrane with 5% skim milk powder at room temperature for 2 hours, and wash 3 times with TBST buffer; Using mouse anti-His tag antibody as positive control and healthy human serum as negative control, add sera from patients with Schistosomiasis haematobium, Schistosomiasis mansoni and Schistosomiasis japonicum patients (diluted with blocking solution 1:100), 4°C Incubate overnight, wash 3 times with TBST buffer; add fluorescently labeled anti-mouse IgG antibody or anti-human IgG antibody (diluted with blocking solution 1:10,000), incubate at 37°C for 1 hour in the dark, wash 3 times with TBST buffer; use Odyssey infrared laser imaging system scanning imaging. The result is as image 3 As sho...

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Abstract

The invention discloses a recombinant fusion protein ShSAP of Schistosoma haematobium and its application in immunodiagnosis of Schistosomiasis. The Schistosoma haematobium recombinant fusion protein ShSAP of the present invention can be mass-produced in large-scale standardized batches through genetic engineering technology, the antigen quality is stable, and the result repeatability is good. The Schistosoma haematobium recombinant fusion protein ShSAP of the invention can be used for the immunodiagnosis of Schistosoma haematobium and Schistosoma mansoni, and has extremely high sensitivity. The Schistosoma haematobium recombinant fusion protein ShSAP of the present invention has a single antigenic component, and the ShSAP-ELISA has excellent specificity for the immunodiagnosis of schistosomiasis, and does not cross-react with sera of patients with other parasitic diseases, and has broad application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a Schistosoma haematobium recombinant fusion protein ShSAP and its application in immunodiagnosis of schistosomiasis. Background technique [0002] Schistosomiasis is an infectious parasitic disease that seriously threatens human health. It is mainly prevalent in 76 countries and regions in Asia, Africa and Latin America. The number of people infected with schistosomiasis exceeds 200 million worldwide. There are 6 main species of schistosomiasis that parasitize the human body, including Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, Schistosoma intercalatum, Schistosoma mekong and Schistosoma malayi, among which the epidemic range of schistosomiasis caused by Schistosoma japonicum, Schistosoma mansoni and Schistosoma egypti The widest and most harmful. Schistosomiasis japonicum is mainly prevalent in the Yangtze River Basin of my country and Southeast Asian cou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/62C12N15/70C12N1/21C07K19/00G01N33/68C12R1/19
CPCC07K14/43559C07K2319/00C12N15/70G01N33/6893G01N2333/43547G01N2800/26
Inventor 刘帅陈启军侯楠朴贤玉
Owner INST OF PATHOGEN BIOLOGY CHINESE ACADEMY OF MEDICAL SCI
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