Separating method of hepatitis B virus
A technology of hepatitis B virus and separation method, applied in the field of hepatitis B virus separation, can solve the problems of inability to effectively block HBV replication and reinfection, lagging research progress, affecting new treatment methods and drug research and development, etc.
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[0006] 1. Different gene fragments of PreS1 were amplified by PCR method, cloned into eukaryotic secretory expression vector pCMV-CFT with Flag tag, and the Flag-PreS1 fusion protein was further isolated and purified.
[0007] 2. Take the normal liver tissue (about 40 g) of the patient undergoing partial hepatic lobectomy, separate hepatocytes according to the two-step collagenase perfusion method reported by Protzer U et al., culture and identify them.
[0008] 3. Fully incubate the purified and expressed Flag-PreS1 fusion protein with primary human hepatocyte (PHH) lysate, the fusion protein and the binding protein complex on the liver cell membrane can pass through the FITC-labeled anti-Flag monoclonal antibody (Sigma) specific binding, the cell-binding activity of the HBV PreS1 fragment expressed in fusion to PHH was detected by flow cytometry.
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