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Schistosoma haematobium recombinant fusion protein ShSAP and application of schistosoma haematobium recombinant fusion protein ShSAP in immunodiagnosis of schistosomiasis

A technology of fusion protein and schistosomiasis, applied in the biological field, can solve problems such as differences, and achieve the effects of good repeatability, high sensitivity and good specificity of results

Inactive Publication Date: 2019-12-13
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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  • Schistosoma haematobium recombinant fusion protein ShSAP and application of schistosoma haematobium recombinant fusion protein ShSAP in immunodiagnosis of schistosomiasis
  • Schistosoma haematobium recombinant fusion protein ShSAP and application of schistosoma haematobium recombinant fusion protein ShSAP in immunodiagnosis of schistosomiasis
  • Schistosoma haematobium recombinant fusion protein ShSAP and application of schistosoma haematobium recombinant fusion protein ShSAP in 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 by using bioinformatics methods. The amino acid sequence of the Schistosoma haematobium ShSAP fusion protein is shown in the sequence table SEQ ID NO.1. The ShSAP fusion protein is activated by the Schistosoma haematobium sphingolipid 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 shown in SEQ ID NO. ...

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 schistosoma haematobium recombinant fusion protein ShSAP and application of the schistosoma haematobium recombinant fusion protein ShSAP in immunodiagnosis of schistosomiasis. The schistosoma haematobium recombinant fusion protein ShSAP can be subjected to large-scale standardized batch production through a genetic engineering technology as well as is stable in antigen quality and good in repeatability. The schistosoma haematobium recombinant fusion protein ShSAP can be both used for immunodiagnosis of schistosomiasis haematodia and schistosomiasis mansoni and has extremely high sensitivity. The schistosoma haematobium recombinant fusion protein ShSAP is single in antigen component, ShSAP-ELISA has very good specificity for immunodiagnosis of the schistosomiasis and does not generate cross reaction with serums of patients with other parasitic diseases, and the schistosoma haematobium recombinant fusion protein ShSAP has a wide application prospect.

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 Applications(China)
IPC IPC(8): C12N15/12C07K19/00C12N15/70C12N1/21G01N33/569C12R1/19
CPCC07K14/44C07K2319/00G01N33/56905
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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