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应用pET28(含T_7lac启动子和His6-tag)质粒,将人γTNFβ融合基因与之组建成pETγLT表达质粒,并转化于E.coliBL21(DE3).本文对pETγLT/ BL21(DE3)工程菌的His6-γTNFβ重组产物进行了诱导表达,分离纯化的研究.结果表明,该工程菌用LB培养基在37℃扩增至OD_(590)为0.5左右,加IPTG(终浓度为1mmol/L)诱导5小时,此时His6-γTNFβ的表达量可占菌体总蛋白的45%.重组产物绝大多数以IBs形式存在于菌体中.工程菌经反复超声破碎、高速离心,得IBs粗制品后,再经含2mol/L脲的缓冲液洗涤可得到相对纯净的IBs.然后用7mol/L脲变性溶解IBs,离心取上清,进行Ni~(2+)-Sepharose 6B柱一步法亲和层析,可得到电泳纯度为95%的His6-γTNFβ,经稀释复性和用凝血酶切去His6-tag的产物,其细胞毒活性为(1.2～2.0)×10~7U/mgp,抗病毒活性为(6.0～6.6)×10~6U/map.
The pETγLT expression plasmid was constructed with pET28 (containing T_7lac promoter and His6-tag) plasmid and transformed into E.coli BL21 (DE3) γTNFβ recombinant product was induced, isolated and purified.The results showed that this engineering strain was amplified with LB medium at 37 ℃ to OD590 of about 0.5, and induced by IPTG (final concentration of 1mmol / L) for 5 hours , At this time His6-γTNFβ expression can account for 45% of the total bacterial protein. Most of the recombinant products exist in the form of IBs in bacteria. Engineering bacteria after repeated ultrasonic disruption, high-speed centrifugation to obtain crude IBs, and then Relatively pure IBs were obtained by washing with buffer containing 2 mol / L urea, IBs were then denatured with 7 mol / L urea, and centrifuged to collect the supernatant for one-step affinity chromatography on Ni2 + Sepharose 6B column. His6-γTNFβ with 95% electrophoretic purity was obtained. The product of His6-tag was diluted and refolded, and its cytotoxicity was (1.2-2.0) × 10-7 U / mgp. The antiviral activity was ( 6.0 ~ 6.6) × 10 ~ 6U / map.