rhIL-6大肠杆菌高效表达的影响因素及其产品中试的研究

研究自构建的重组人白细胞介素－ ６（ｒｈＩＬ－６）基因工程菌株高效表达的影响因素及其ｒｈＩＬ－６产品中试工艺。方法：①将ｒｈＬ－６－ＰＢＶ重组质粒转化至ＲＲＩ、ＪＭ１０３和 ＤＨ５ ａ不同宿主菌中，在Ｍ９ＣＡ和 ＬＢ培养基中，４２℃诱导培养不同时间（３～６ ｈ），观测 ｒｈＩＬ－６不同表达效率。②采用 １０ Ｌ发酵罐诱导培养，经破菌－洗包－溶包初步纯化后，再经三步柱层析纯化，提取ｒｈＩＬ－６。结果：①ｒｈＩＬ－６在ＤＨ５ ａ大肠杆菌中（ＳＤＨ－９４５株）表达效率高；可达 ３９％，用 Ｍ９ＣＡ培基，４２℃诱导培养 ５ｈ可使表达效率提高到４１％，②ＳＤＨ－９４５菌株在５批 １０Ｌ Ｍ９ＣＡ发酵诱导培养 ５ ｈ，ｒｈＩＬ－６表达效率可达 ３５． ８７±２． ６５％。经初步纯化后，ｒｈＩＬ－６的纯度达 ７４． ８７±３． ６８％，再经三步柱层析后，纯度可达 ９５％以上，比活性达 ４ × １０８ Ｕ／ｍｇ，总得率稍低可达 ２３．１％．结论：①ＳＤＨ－９４５工程菌株在 Ｍ９ＣＡ培养基巾，４２℃诱导培养 ５ ｈ，ｒｈＩＬ－６表达效率最佳。②１０ Ｌ发酵和纯化工艺可获高效价、高比活性、高纯度的ｒｈＩＬ－６合格生物制品。

Study on the Influential Factors of High Expression of rhIL-6 E. coli and Its Middle Scale of Products

Purpose: To study influential factors of the high expression of rhlL-6 E. coli constructed by our department and its middle scale of product. Methods: ① We transformate rhIL-pBV plasmid into different hosts of RRI, JM103 and DH5 a. and observe their different expression rate under the conditions of different media (M9 CA or LB) and different time of inducing cultivation (three-six hours) at 42℃ ②After inducing cultivation in 10L fermentation tank, carry on primary purification through breaking E. coli-Washing inclusion bodies-soluting inclusion bodies, and further purification by sephadex G50 - DEAE-Fibre column-sephacry S 200 to get rhIL-6. Results: ①The expression rate of rhIL-6 is higher in DH5 a E. coli (SDH-945), and reach 39%, while using M9 CA media for five hours at 42℃, it can improve to 41 %. ②SDH-945 carry 5 lots of inducing cultivation for five hours in the 10L fermentation tank, its expression rate can reach 35. 87±2. 65 %. After primary purification, the purity of rhIL-6 reach 74. 87 ± 3. 68%, and after further three steps purifying, the purity reach 95%, the contrast activity reaches 4 × 108 U/mg, the total output is lower and reach 23. 1 %. Conclusion: ①The expression rate of rhlL-6 is best by cultivating SDH-945 under the conditions that at 42 ℃ in the media of M9CA for 5 hours. ②This technic of 10L fermentation tank and purity can get qualified product of rhIL-6 of high efficiency. high contrast activity and high purity.