Study And Application On The Affinity Chromatography Technology

Affinity chromatography is a method to purify proteins, which possesses the property of high selection and separation, as well as larger load. The target protein with purification of more than a thousand times and a high activity could be obtained from the mixture using only one step in affinity chromatography. Affinity ligand is a key in this separation technique, which is fixed to the hydrophilic solid-phase media to purify the target protein through the specific recognition.After Sephadex G-25 polymerbeads were activated by using epichlorohydrin, hemin was binded with them to prepare an immobilized hemin affinity chromatography column, which was used to prepare antioxidant proteins from ginkgo and soybean. Equilibrated with deionized water and eluated with pH 3.6,0.2 mol/L NaAc-HAc buffer, those proteins obtained from this columne were demonstrated to possess high antioxidative activity(AA) after measurement with linoleic acid-potassium thiocyanate. AA of crude proteins of ginkgo was 7.92% and two polypeptides of them were obtained with AA 17.45% after purification. In soybean, several polypeptides with AA 30.72% were obtained from the crude proteins with AA 10.52%. This study offered a novel, rapid and effective method for preparation of antioxidant proteins from ginkgo and soybean.After Sepharose-4B polymerbeads were activated by using epichlorohydrin, purified swine fever virus obtained from the culture of PK-cells were binded with them as a ligand to prepare an immunoaffinity chromatography column, which was used to prepare rapidly and identify antibody against classical swine fever virus from high immune pig serum. SDS-PAGE and ELISA were used to determine those proteins purified by this column. The proteins isolated by this column from crude high immune pig serum were demonstrated to be the pure antibodies and possess normal activity and specificity. The extraction efficiency of the antibody was 2.45% of total proteins. This study offers a novel, rapid and effective method for preparation of pure antibodies against classical swine fever virus from high immune pig serum.