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本实验研究了不同温度、不同KCNS浓度、不同PH及不同盐析次数对人脐血白细胞干扰素(下称人脐血IFN)的活件、纯度和回收的影响;观察了人脐血IFN与蓝色葡聚糖的亲和能力及其在Ccn A-Sepharose 4B柱和蓝葡聚糖2000-Sepharose 4B(BDS)柱上的层析行为;建立了简易可行的IFN纯化系统;测定了人脐血IFN的分子量;並提出了人脐血IFN是一组糖基隐蔽在分子内部的糖蛋白质的设想 建立的IFN纯化系统包括两步:第一步为“粗提”,系采用KCNS-酒精法;第二步为“精提”,系采用BDS柱亲和层析法。实验结果表明:沉淀粗制干扰素的最适条件是加入0.5M KCNS、调至pH4.0;析出干扰素的最佳pH是5.5～7.2;KCNS盐析一次与两次结果大致相同;冰酒精保冷普通离心机离心,对酒精溶液中的IFN活性无明显影响。粗制干扰素经第一步纯化,活性回收70％左右,比活性约为10~6国际单位mg蛋白~(-1)。达到了临床级IFN的国际标准,可供临床试用;再经第二步,用BDS柱纯化,以0.4 M NaCl和0.8 M NaCl液分段洗脱,IFN活性回收40％以上,最高活性组份的纯度达10~7国际单位mg蛋白~(-1)。分析性SDS-PAGE上呈现两条蛋白质区带。 实验结果还表明,人脐血干扰素对SDS、核酸酶和pH 2稳定;对蛋白酶敏感;具有明显的种属特异性;SDS-PAGE凝胶切片活性测定证明分子量在18.3K～24
In this study, the effects of different temperature, different KCNS concentration, different pH and different salting-out times on the activity, purity and recovery of human umbilical cord blood leukocyte interferon (IFN-γ) Affinity of blue dextran and its chromatographic behavior on Ccn A-Sepharose 4B column and Blue Dextran 2000-Sepharose 4B (BDS) column; a simple and feasible IFN purification system was established; Blood IFN and molecular weight; and proposed that human umbilical cord blood IFN is a group of sugar-based concealment within the molecule of the glycoprotein hypothesis established IFN purification system consists of two steps: the first step is “crude extract”, the Department of KCNS-alcohol method ; The second step is “refined extract”, the Department of BDS column affinity chromatography. The experimental results showed that the optimum conditions for precipitating interferon were as follows: adding 0.5M KCNS to pH4.0; the optimum pH for precipitation of interferon was 5.5-7.2; the results of KCNS salting were almost the same as those of twice; Centrifuge Centrifuge centrifuge, no significant effect on the IFN activity in alcohol solution. Crude interferon purified by the first step, the activity of about 70% recovery, the specific activity of about 10 ~ 6 International units of mg protein ~ (-1). Reached the clinical level of IFN international standards for clinical trial; then the second step, with BDS column purification, 0.4 M NaCl and 0.8 M NaCl elution fractionation, IFN activity recovery of more than 40%, the highest active component The purity of 10 ~ 7 International units mg protein ~ (-1). Two protein bands appear on analytical SDS-PAGE. The results also showed that human umbilical cord blood interferon on SDS, nuclease and pH 2 stable; sensitive to protease; has obvious species-specific; SDS-PAGE gel section activity determination of molecular weight 18.3K ~ 24