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Schistosoma mansoni recombinant fusion protein SmSAP and application thereof to schistosomiasis immunodiagnosis

A technology of Schistosoma mansoni and fusion protein, which can be applied in the biological field and can solve problems such as differences

Active Publication Date: 2021-05-11
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 Schistosoma japonicum protein and homologous protein of Schistosoma mansoni, the existing immunodiagnostic methods of Schistosoma japonicum cannot fully meet the needs of immunodiagnosis of Schistosoma mansoni

Method used

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  • Schistosoma mansoni recombinant fusion protein SmSAP and application thereof to schistosomiasis immunodiagnosis
  • Schistosoma mansoni recombinant fusion protein SmSAP and application thereof to schistosomiasis immunodiagnosis
  • Schistosoma mansoni recombinant fusion protein SmSAP and application thereof to schistosomiasis immunodiagnosis

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Schistosoma mansoni SmSAP fusion protein gene synthesis and prokaryotic expression vector construction

[0028] The SmSAP gene encoded by the whole genome of Schistosoma mansoni is predicted by bioinformatics methods. The amino acid sequence of the SmSAP fusion protein of Schistosoma mansoni is shown in the sequence table SEQ ID NO.1. The SmSAP fusion protein is composed of Schistosoma mansoni with the signal peptide sequence removed Saposin-like protein 1 (SmSAP1) and Schistosoma mansoni saposin-like protein 2 (SmSAP2), the amino acid sequences of SmSAP1 protein and SmSAP2 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 SmSAP fusion protein coding gene was designed, and then the SmSAP fusion gene of Schistosoma mansoni was prepared by gene synthesis technology, and its nucleotide sequence was shown in SEQ ID NO.4 in the sequence table. Nde I and Xho I restriction sites were d...

Embodiment 2

[0030] Example 2 Prokaryotic expression and purification of Schistosoma mansoni SmSAP fusion protein

[0031] Transform the pET28a-SmSAP 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 prote...

Embodiment 3

[0035] Example 3 Antigenicity Detection of Schistosoma mansoni SmSAP Recombinant Protein

[0036] Take 200ng of SmSAP 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 serum from patients with Schistosomiasis mansoni, Schistosomiasis haematobium 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 shown,...

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Abstract

The invention discloses a schistosoma mansoni recombinant fusion protein SmSAP and an application thereof to schistosomiasis immunodiagnosis. The schistosoma mansoni recombinant fusion protein SmSAP disclosed by the invention can be subjected to large-scale standardized batch production through a genetic engineering technology, the antigen quality is stable, and the result repeatability is good. The schistosoma mansoni recombinant fusion protein SmSAP disclosed by the invention not only can be used for immunodiagnosis of schistosoma mansoni, but also can be used for immunodiagnosis of schistosoma egyptiacum, and has extremely high sensitivity. The schistosoma mansoni recombinant fusion protein SmSAP is single in antigen component, SmSAP-ELISA has excellent specificity on schistosomiasis immunodiagnosis, cross reaction with serum of other parasitic disease patients is avoided, and the application prospect is wide.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant fusion protein SmSAP of Schistosoma mansoni 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 coun...

Claims

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Application Information

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IPC IPC(8): C12N15/62C07K19/00C12N15/70C12N1/21G01N33/68G01N33/58G01N33/545G01N33/543C12R1/19
CPCC07K14/43559C07K2319/00C12N15/70G01N33/54393G01N33/545G01N33/581G01N33/6854G01N2333/43547
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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