A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling ...A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.展开更多
Objective: To obtain pure human monoclonal antibody (mAb) Fab fragments against HBsAg with good biological activity by genetic engineering technology. Methods: The specific anti-HBsAg phagemid was selected from establ...Objective: To obtain pure human monoclonal antibody (mAb) Fab fragments against HBsAg with good biological activity by genetic engineering technology. Methods: The specific anti-HBsAg phagemid was selected from established combinatorial library and transfected into E. coil XL1-blue. Its expression was induced by isopropyl β-D- thiogalactopyranoside (IPTG). The crude Fab supernatant was obtained after E. coli cells were frozen at - 20℃ and thawed repeatedly along with centrifugation. The goat anti-human IgG Fab was prepared by immunizing the goat with purified human IaG Fab. The affinity chromatography column with goat anti-human IgG Fab and GammaBind Plus Sepharose was obtained. The crude Fab super- natant was purified with affinity chromatography and the purity was assessed with SDS-PAGE and Western-blot, and biological activity was evaluated by Dot-blot test. Results: SDS-PAGE of the purified Fab displayed a side band, verified to be the Fab band by Western-blot test. Dot-blot test demonstrated that the purified Fab fragments posses good affinity to HBsAg. Conclusion: The success in purification of human anti-HBsAg Fab fragments with good biological activity makes it possible to be used as future therapeutic agents.展开更多
对利用金属离子亲和层析纯化重组类人胶原蛋白过程中使用的金属离子进行了比较,从而对分离纯化的条件进行优化。在相同实验条件下,用4种金属离子柱分离纯化目的蛋白。结果显示,经4种金属离子柱纯化后镍柱的总蛋白收获率最高,铜柱与锌柱...对利用金属离子亲和层析纯化重组类人胶原蛋白过程中使用的金属离子进行了比较,从而对分离纯化的条件进行优化。在相同实验条件下,用4种金属离子柱分离纯化目的蛋白。结果显示,经4种金属离子柱纯化后镍柱的总蛋白收获率最高,铜柱与锌柱居中,钙柱最低;而柱保留时间则为锌柱最高(12.38 m in),钙柱最低(8.25 m in);钙柱洗脱时所需咪唑解离初始浓度最低(100 mmol/L),锌柱最高(200 mmol/L);对SDS-PAGE电泳图进行分析得纯化后类人胶原蛋白的纯度分别为:镍柱82.8%,铜柱83.4%,锌柱96.2%,钙柱94.3%。由此可见锌柱对目标蛋白的亲和力最高,且纯化后类人胶原蛋白的纯度也最高。因此,确定锌离子作为亲和层析纯化重组类人胶原蛋白的金属离子。展开更多
文摘A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.
文摘Objective: To obtain pure human monoclonal antibody (mAb) Fab fragments against HBsAg with good biological activity by genetic engineering technology. Methods: The specific anti-HBsAg phagemid was selected from established combinatorial library and transfected into E. coil XL1-blue. Its expression was induced by isopropyl β-D- thiogalactopyranoside (IPTG). The crude Fab supernatant was obtained after E. coli cells were frozen at - 20℃ and thawed repeatedly along with centrifugation. The goat anti-human IgG Fab was prepared by immunizing the goat with purified human IaG Fab. The affinity chromatography column with goat anti-human IgG Fab and GammaBind Plus Sepharose was obtained. The crude Fab super- natant was purified with affinity chromatography and the purity was assessed with SDS-PAGE and Western-blot, and biological activity was evaluated by Dot-blot test. Results: SDS-PAGE of the purified Fab displayed a side band, verified to be the Fab band by Western-blot test. Dot-blot test demonstrated that the purified Fab fragments posses good affinity to HBsAg. Conclusion: The success in purification of human anti-HBsAg Fab fragments with good biological activity makes it possible to be used as future therapeutic agents.
文摘对利用金属离子亲和层析纯化重组类人胶原蛋白过程中使用的金属离子进行了比较,从而对分离纯化的条件进行优化。在相同实验条件下,用4种金属离子柱分离纯化目的蛋白。结果显示,经4种金属离子柱纯化后镍柱的总蛋白收获率最高,铜柱与锌柱居中,钙柱最低;而柱保留时间则为锌柱最高(12.38 m in),钙柱最低(8.25 m in);钙柱洗脱时所需咪唑解离初始浓度最低(100 mmol/L),锌柱最高(200 mmol/L);对SDS-PAGE电泳图进行分析得纯化后类人胶原蛋白的纯度分别为:镍柱82.8%,铜柱83.4%,锌柱96.2%,钙柱94.3%。由此可见锌柱对目标蛋白的亲和力最高,且纯化后类人胶原蛋白的纯度也最高。因此,确定锌离子作为亲和层析纯化重组类人胶原蛋白的金属离子。
