STUDIES ON PURIFICATION AND SOME PROPERTIES OF LEUKOCYTE INTERFERON FROM HUMAN UMBILICAL CORD BLOOD
- VernacularTitle:人脐血白细胞干扰素的纯化及性质研究
- Author:
Zhongtian QI
- Publication Type:Journal Article
- From:
Academic Journal of Second Military Medical University
1983;0(S1):-
- CountryChina
- Language:Chinese
-
Abstract:
By studies of primary influencing factors in interferon ( IFN ) purification as well as affinity of IFN for different ligands, a purification system for leukocyte IFN from human umbilical cord blood was preliminarily established, and the properties of this IFN were studied.The established purification system includes two steps. The first step may be called "rough purification" using potassium thiocyanate (KCNS)-alcohol method. The second step "fine purification" using Blue Dextran-sepharose 4B(BDS) column affinity chromatography. The effects of different temperature, KCNS concentration, pH and the time(s) of salting out with KCNS on the activity, purity and recovery of the IFN were studied. Moreover, the affinity of the IFN for Blue Dextran and the chromatographic behavior of it on ConA-sepharose 4B column and BDS column were also observed.The experimental results showed that optimal conditions for precipitating the IFN from the crude IFN solution were to add KCNS to 0.5M, and then adjust pH to 4.0. The optimum pH values in separating out the IFN from acid alcohol-interferon were 5.5 to 7.2. The results between the salting out of once and twice by KCNS were not significantly different. Centrifugation with centrifuge maintaining lower temperature with cold alcohol might be used instead of refrigerated centifuge during purification. The IFN can be purified to about 106 international units per mg of protein with about 70% recovery on the first step of purification. On the second step of purification, the specific activity of IFN in the collecting fraction with maximum activity was increased up to about 107 international units per mg of protein and the recovery of activity was over 40%.Furthermore, the present findings indicated that the IFN from human umbilical cord blood was resistant to treatment with Sodium Dodeyl Sulfate(SDS), nucleolytic enzymes and pH 2.0; but sensitive to proteolytic enzymes. It was proved that the IFN had clearly species specificity. The determination of antiviral activity titer in SDS-PAGE gel slices documented that molecular weights of the IFN was about 18.3k to 24.3k. The IFN could be bound to Blue Dextran, but almost not to ConA. It indicates that IFN from human umbilical cord blood belongs in human IFN-? (Le) and is probably a glycoprotein that its carbohydrate moieties had hidden within IFN molecules.The present experiments may offer the possibility in purifying the IFN for clinical use and be helpful in revealing the essence of IFN from human umbilical cord blood. The purification method discribed in the paper will render some valuable reference to other types of IFN.
