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
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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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