The fusion gene of BPI 23 and human Fcγ1 was obtained by PCR method,and the expression plasmid was constructed to express recombinant BPI 23 \|Fcγ1 fusion protein in CHO cells.After transfection with the plasmid and...The fusion gene of BPI 23 and human Fcγ1 was obtained by PCR method,and the expression plasmid was constructed to express recombinant BPI 23 \|Fcγ1 fusion protein in CHO cells.After transfection with the plasmid and selection by methotrexate,the cell lines expressing the fusion protein were obtained.The recombinant protein was purified using cation\|exchange chromatography and its bioactivity was proved with bactericidal assays.展开更多
目的:通过优化毕赤酵母的表达参数,为提高BPI23-haFGF融合蛋白在毕赤酵母中的表达量创造条件。方法:采用小型摇瓶培养系统,对重组毕赤酵母菌X-33/pPICZαA-BPI23-haFGF在含有蛋白酶抑制剂PMSF以及不同诱导时间、培养温度、甲醇浓度及培...目的:通过优化毕赤酵母的表达参数,为提高BPI23-haFGF融合蛋白在毕赤酵母中的表达量创造条件。方法:采用小型摇瓶培养系统,对重组毕赤酵母菌X-33/pPICZαA-BPI23-haFGF在含有蛋白酶抑制剂PMSF以及不同诱导时间、培养温度、甲醇浓度及培养液pH值条件下进行表达,将表达上清进行SDS-PAGE电泳,并用Quantity one Volume Rect Tool和Dentity tool进行分析。结果:在诱导培养基中加入0.5mmol/L PMSF后,BPI23-haFGF融合蛋白降解得到缓解,诱导表达96h表达量达到高峰;在培养温度28℃,甲醇浓度为1.0%,培养液pH 6.0时,BPI23-haFGF表达量最高,含量比优化前提高了91.6%。结论:该研究为BPI23-haFGF融合蛋白后期的大规模发酵及深入研究其生物学功能奠定了基础。展开更多
文摘The fusion gene of BPI 23 and human Fcγ1 was obtained by PCR method,and the expression plasmid was constructed to express recombinant BPI 23 \|Fcγ1 fusion protein in CHO cells.After transfection with the plasmid and selection by methotrexate,the cell lines expressing the fusion protein were obtained.The recombinant protein was purified using cation\|exchange chromatography and its bioactivity was proved with bactericidal assays.
文摘目的:通过优化毕赤酵母的表达参数,为提高BPI23-haFGF融合蛋白在毕赤酵母中的表达量创造条件。方法:采用小型摇瓶培养系统,对重组毕赤酵母菌X-33/pPICZαA-BPI23-haFGF在含有蛋白酶抑制剂PMSF以及不同诱导时间、培养温度、甲醇浓度及培养液pH值条件下进行表达,将表达上清进行SDS-PAGE电泳,并用Quantity one Volume Rect Tool和Dentity tool进行分析。结果:在诱导培养基中加入0.5mmol/L PMSF后,BPI23-haFGF融合蛋白降解得到缓解,诱导表达96h表达量达到高峰;在培养温度28℃,甲醇浓度为1.0%,培养液pH 6.0时,BPI23-haFGF表达量最高,含量比优化前提高了91.6%。结论:该研究为BPI23-haFGF融合蛋白后期的大规模发酵及深入研究其生物学功能奠定了基础。
