Preparation and application of hepatitis C virus recombinant protein
A virus and protein technology, applied in the direction of anti-viral immunoglobulin, application, viral peptide, etc., can solve the problem of not being broad-spectrum
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[0221] Example 1 Expression of sE2 in Drosophila S2 cells
[0222] The recombinant plasmid used to express HCVsE2 protein in Drosophila S2 cells such as figure 1 As shown in A, carrying the sE2 wild-type coding gene or codon-optimized gene, the target protein encoded is the extracellular region of E2 protein (including amino acids 384-661). The vectors expressing wild-type sE2 encoding genes or codon-optimized genes were transiently transfected into Drosophila S2 cells, and 2×10 were taken out 48h after transfection 6 Cells were added with chromium chloride at a final concentration of 5μM to induce expression. After 72 hours, the supernatant was collected and the detection showed that the optimized sE2 expression was significantly enhanced ( figure 1 B). Therefore, the inventors selected codon-optimized expression vectors for subsequent experiments. The pMT-Bip / sE2opti / His and pCoBlast were co-transfected into Drosophila S2 cells. After blasticidal screening, the monoclonal cell ...
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[0224] Example 2 Characteristic analysis of sE2 protein
[0225] The sE2 protein was detected and purified by Western blot with His monoclonal antibody and E2 monoclonal antibody (AP33), respectively, and both produced a positive band of about 46kDa, indicating that the protein is indeed the target sE2 protein ( figure 2 A). The sE2 protein was treated with PNGaseF to deglycosylate and then tested with the same antibody. The position of the positive band was found to be about 34kDa ( figure 2 A), suggesting that the sE2 protein expressed by S2 insect cells has glycosylation modification.
[0226] Through ELISA detection, sE2 can well bind to the monoclonal antibody AR3A that recognizes discontinuous conformational epitopes, and its ELISA reading is positively correlated with the monoclonal antibody concentration; although sE2 binds to the monoclonal antibody AP33 that recognizes linear epitopes to a certain extent, the response is not strong ( figure 2 B). This result suggests t...
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[0230] Example 3 sE2 can specifically detect the serum of HCV infected patients
[0231] In order to determine whether sE2 can specifically bind to HCV-specific antibodies in HCV-infected persons, the present inventors coated the ELISA plate with sE2 protein, and respectively used diluted healthy human serum and two subtypes (1b and 2a) of hepatitis C patient serum Incubate, and then detect specific IgG antibodies that bind to sE2.
[0232] The results showed that compared with healthy human serum, patient serum produced higher binding activity, and the average OD value at 1:20000 dilution was still as high as 0.5-1.0 ( image 3 ), indicating that sE2 can efficiently, specifically and broadly bind the anti-E2 antibodies in the serum of infected patients. Therefore, sE2 can be used to develop a simple and rapid diagnostic kit for HCV infection.
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