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Examples
Experimental program
Comparison scheme
Effect test
Embodiment 1
[0037] 1. The crudely purified recombinant herpes simplex virus supernatant was filtered at room temperature using a serotonin TuffrynR polysulfone membrane capsule filter device to remove any cells and cell debris.
[0038] 2. Use a stirred cell ultrafiltration device for ultrafiltration, and an Omega polyethersulfone membrane disc filter for concentration.
[0039] 3. During osmosis, use heparin affinity adsorption buffer to enrich and intercept the virus, repeat the same pattern for 3 times, and use 100mL of buffer each time.
[0040] 4. Filter the concentrated virus supernatant through a 0.45um GHP Acrodisc filter.
[0041] 5. Add tentacle-type FractogelR EMD Heparin (S), Fractogel EMD DEAE (M), and Fractogel EMD SO 3 - (M) gels (Merck, Darmstadt, Germany) were packed into HR 5 / 5 columns to a final volume of 1 mL and equilibrated in buffer: 20 mM HEPES buffer, pH 7.5, under salt-free conditions.
[0042] 6. Load 0.5mL of the sample filtered by the GHP Acrodisc membrane i...
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PUM
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Abstract
The invention relates to a heparin affinity chromatography method of recombinant herpes simplex virus. The method comprises: filtering out cell debris from the virus supernatant, and concentrating by using a polyethersulfone membrane disc type filter; carrying out cryopreservation on the concentrated solution in a -80 DEG C refrigerator; loading heparin into a HR 5/5 column, thawing the concentrated sample, and then loading the thawed sample into the column; and carrying out elution, collecting three peak value eluents, and carrying out cryopreservation at a temperature of -80 DEG C or in liquid nitrogen. The method of the present invention has the following advantages that: the molecular characteristic that the heparin affinity chromatography column has a large number of negatively charged sulfate groups is used, the negatively charged sulfate groups can be adopted as the affinity ligand in protein and virus purification so as to selectively bind with the protein molecules and other molecules on the virus surface, and the salt ions with different concentrations are used to carry out gradient elution so as to achieve the separate virus elution purpose.
Description
technical field [0001] The invention relates to the technical field of virus purification, more specifically to heparin affinity chromatography of recombinant herpes simplex virus, and belongs to the field of biomedicine. Background technique [0002] Heparin is a highly sulfated glycosaminoglycan with the highest negative charge density among known biomolecular materials and is widely used in biomedical fields. However, the existing purification methods of heparin are difficult to industrialized large-scale production. Contents of the invention [0003] The purpose of the present invention is to provide an affinity chromatography method for recombinant herpes simplex virus, which is mainly divided into preliminary purification and concentration and high performance liquid chromatography, aiming at the current situation that the existing heparin purification method is difficult for industrialized large-scale production. Two fractions were purified. [0004] The present ...
Claims
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