人源抗-HBs Fab优化表达条件的初步探讨

目的：利用含重组质粒pBAD/HBs Fab的Top10大肠杆菌，优化表达人源抗－HBs Fab。方法：选取含pBAD/HBs Fab载体的Top10大肠杆菌单个克隆分级培养，将制备的二级种子液接种于摇瓶和KLF2000发酵罐中，摇瓶培养表达采用不同的菌体诱导起始密度、诱导剂浓度、诱导表达温度和诱导表达时间。发酵罐培养表达采用不同的培养基和菌体诱导起始密度。诱导表达完毕后，纯化蛋白，比较表达量，并鉴定所获蛋白的抗原性和生物学活性。结果：用摇瓶优化表达后，Fab蛋白占菌体总蛋白的6％，为0.8%mg/g菌体，利用发酵培养可进一步增加菌量，使蛋白表达量达80mg/L。所获蛋白经鉴定显示有较好的生物学活性。结论：用重组质粒pBAD/HBsFab,Top10表达系统，可得到80mg/L的具有较好生物学活性的人源抗－HBs Fab蛋白，为批量生产作了准备。

Studies on the optimal expression condition of human anti-HBs Fab fragment

Objective To obtain an optimal conditions to produce huma n anti HBs Fab by using pBAD/HBsFab,Top10 expression system.Metho ds E.coli Top10 containing recombinant plasmid pBAD/HBs Fab was grown in LB m edium by two stages ,then was inoculated into spinner flask and fermenter. T he c onditions including cell density of induction, concentration of arabinose, expre ssion temperature, and expression time in spinner flask were optimized. The opti mal conditions were applied to fermentation using semi defined and defined medi um ,then the authors optimized the cell density of induction in fermenter. After culture and expression processes, Fab protein was purified, and the antigenicity and bin ding activity to HBsAg were verified.Results The optimal condi tions were found and cell concentration thus obtained is 96 g wet bacteria p er l iter. The Fab protein is about 6% of total protein of the host .That is 80 mg pe r liter.Conclusion Stable optimal conditions were obtained to p roduce Fab protein. The Fab protein was produced in the form of soluble biologic ally active protein. The strategy is available in gene engineering technology. [