重组人白细胞介素6的制备

Preparation of Recombinant Human Interleukin-6

我们采用本院基础医学研究所组建的ＩＬ—６工程菌Ｅ．ｃｏｉｌＤＨ５ａ（ｐＢＶ—ｈｌＬ－６），在选定的培养基及ｐＨ值下，采用３０升发酵罐进一步观察了工程菌的生长和ｒＩＬ－６的表达，确定了发酵工艺条件。在此条件下连续进行了三批次的培养试验。结果表明，工程首的生长密度达到２．５１±０．０２ｇ［干重］／Ｌ［发酵液］，ｒＩＬ—６产率为１８２．４±２．０ｍｇ／ｇ［干重］。ｒＩＬ—６以包涵体形式表达于大肠杆菌细胞中，破菌后选用非离子型去垢剂或尿素等变性剂提取包涵体中的杂蛋白，可使ｒＩＬ—６的纯度达到７０．１±１．３％，收率为７１．９±１．９％。洗涤后的包涵体，经过凝胶柱纯化和复性，ｒＩＬ—６纯度达到９５％以上，柱纯化的收率为７２．３±０．９％；采用依赖ＩＬ—６，小鼠杂交瘤细胞系７ＴＤ１及ＭＴＴ比色法测定生物活性，ｒＩＬ—６比活性达２×１０８Ｕ／ｍｇ。

We have adopted recombinant IL - 6 (rIL - 6 ) producing E. coli DH5a (pBV - hIL) and observed the growth and expression of rIl-6 in 30 liters fermentor under the selected composition of medium and pH value. The fermentative technological conditions were determined. The results of three batches of experiment under the conditions showed that the cell density of recombiant bacteria was 2. 51 ±0. 02g [dry weight]/L[fermcnted liquor]and the yield of rIL-6 was 182. 4±2. 0mg/g [dry weight]. rIL-6 is expressed in form of inclusion bodies in cells. Impurified proteins in inclusion bodies were extracted by nonion-detergent or urea or the other denaturant and the purity of rIL-6 can reach 70. 1± 1. 3% and the yield is 71. 9± 1. 9% The washed inclusion bodies were purified through gel filtration chromatography and renatured. The purity of rIL-6reached above 95 % and the yield was 72. 3±0. 9%. The specific activity of rIL-6 was 2 × 108U/mg which detected by using the IL-6-dependent mouse - mouse hybrid Cell line 7TD1 and MTT colorimetric assay.