密码替换后人促红素基因在大肠杆菌中的高效表达及工程菌的发酵

目的　研究人工合成的hEPO基因在大肠杆菌中高效表达及工程菌高密度发酵条件。方法　将hEPO基因中大部分大肠杆菌稀有密码子替换成使用频率较高的密码子 ,人工合成hEPO全基因 ,然后将其插入表达载体PBV2 2 0中 ,转化大肠杆菌DH5α ,挑选构建正确的PBV2 2 0 hEPO DH5α菌落 ,经升温诱导 ,SDS- PAGE和Westernblot鉴定表达产物 ;观察改变培养基、培养时间、pH及溶氧量等条件对表达产物的影响。结果　经酶切分析 ,DNA测序鉴定 ,人工合成的hEPO基因已正确构建到表达载体中。通过高密度发酵培养 ,最终菌体密度达A6 0 0 =30 (相当于菌体 35g L) ,hEPO表达量达 30 %左右。结论　人工合成的hEPO基因在大肠杆菌中得到高效表达 ,并确定了该工程菌高密度发酵的适宜条件。

High Expression of Human Erythropoietin (EPO) Gene after Coden Replacement in E. coli and Optimization of Fermentation Procedure

Objective To explore the condition for high e xpre ssion of synthetic human erythropoietin (hEPO) in E.coli and for high densit y fermentation.Methods Replace most of the rare codens of E.c oli in hEPO gene with high frequency ones and synthesize the full-length of h EPO gene.Insert the synthetic gene into expression vector PBV220,and transform t he recombinant plasmid to E.coli DH5α for expression under induction of tem perature.Identify the expressed product by SDS-PAGE and Western blot.Meanwhile, the influence of various conditions,such as medium,time for incubation,pH value and dissolved oxygen (DO) concentration on the expressed product,were studied. Results Restriction analysis and DNA sequencing proved that the s ynthetic hEPO gene was correctly inserted into the expression vector.The final b acterial density (A 600) after high density fermentation under an optimal c ondition (42℃,3 h,DO 20%) reached 30 (equivalent to 35 g/L of bacteria),and the expressed hEPO contained about 30% of total somatic protein.Conclusion The synthetic hEPO gene was highly expressed in E.coli,and the con dition for high density fermentation of the recombinant E.coli strain was op timized.