人白细胞介素-12的纯化及鉴定

目的]利用人白细胞介素-12(human interleukin 12,hIL-12)转基因马铃薯,探讨hIL-12的纯化条件。方法]提取转基因马铃薯中的总蛋白,hIL-12的分离纯化步骤包括超滤、MKF-AIW阴离子交换层析、MKF-CIC阳离子交换层析。在阴离子交换层析吸附过程中采用试管法选择最佳吸附pH值,选择洗脱过程最佳洗脱pH值;选择阳离子交换层析洗脱过程最佳洗脱pH值;ELISA法测定各纯化阶段收集的上清液或洗脱峰中的hIL-12的含量;非还原SDS-PAGE电泳鉴定蛋白纯度;Western Blot检测纯化的hIL-12。结果]阴离子交换层析吸附过程最佳吸附pH值为7.5,洗脱过程最佳洗脱pH值为7,阳离子交换层析洗脱过程最佳洗脱pH值为6.5。纯化的hIL-12经非还原SDS-PAGE分析蛋白纯度为91%。Western Blot分析结果表明,在40 kD处有特异的蛋白条带出现。结论]hIL-12转基因马铃薯经总蛋白提取,超滤,阴离子交换层析,阳离子交换层析纯化,获得了高纯度的hIL-12。

Purification and Identification of Human Interleukin 12

Objective] The study aims to discuss purification conditions by using human interleukin 12(hIL-12) from transgenic potatoes.Methods] Total protein is extracted from transgenic potatoes and concentrated solution is purified as follow: ultrafiltration,MKF-AIW anion exchange chromatography and MKF-CIC cation exchange chromatography.Best adsorption pH value and best elution pH value are selected in adsorption and elution process of anion exchange chromatography,respectively.Best elution pH value is selected in elution process of cation exchange chromatography.The content of hIL-12 is assayed in supernatant or eluting peak of various purification phases by ELISA.The purity is measured by non-reduced SDS-PAGE.Purified hIL-12 has been tested by Western Blot.Result] Best adsorption pH value and best elution pH value are 7.5 and 7 in adsorption and elution process of anion exchange chromatography,respectively.Best elution pH value is 6.5 in elution process of cation exchange chromatography.The purity of hIL-12 is 91% by non-reduced SDS-PAGE.Western Blot analysis shows that the specificity protein band presented in 40 kDa.Conclusion] High purity of hIL-12 has been obtained through the process which includes: extracting total protein from transgenic potato,ultrafiltration,anion exchange chromatography and cation exchange chromatography.