肝癌相关抗原SMP-30基因工程菌培养条件的优化

THE OPTIMAL CULTURE CONDITIONS FOR PREPARING RECOMBINANT HUMAN HEPATOCELLULAR CARCINOMA-ASSOCIATED ANTIGEN SMP-30 EXPRESSED IN ENGINEERING E.COLI

目的：对重组人肝癌相关抗原SMP-30基因工程菌的培养条件进行研究,确定最佳培养方案以提高基因工程蛋白表达产量。方法：通过SDS-PAGE蛋白质电泳和电泳图谱扫描来分析诱导剂浓度、诱导起始时间、诱导时间长短、培养温度和培养基成分对基因工程菌TB1表达肝癌相关抗原SMP-30的影响。结果：当工程菌培养条件合适,即在吸光度值A600为0.5左右时加入IPTG至终浓度0.5mmol/L,30℃培养7h,其表达的SMP-30融合蛋白可占细体总蛋白量的46.1%。将工程菌培养温度从37℃降到30℃时,表达蛋白质的可溶性部分可提高10%-15%。结论：通过对重组人肝癌相关抗原SMP-30基因工程菌的培养条件进行优化,获得较满意的应用工程菌表达肝癌相关抗原SMP-30的培养条件,为基因工程下游技术奠定了基础。

Objective： To gain the best recombinant protein expression for generation of human hepatocellular carcinoma-associated antigen SMP-30, the culture conditions of the genetic engineering E. coli （pMAL- C2-SMP-30/TB1） were investigated. Methods： Some parameters involved in the relative amounts of expressed protein were displayed and compared by using SDS-PAGE and Electrophoresis Scanning Software. The concerned factors were concentration of IPTG, the time point of induction, the time course of induction, the temperature and the components of culture medium. Results： Expression level of SMP-30 fusion proteins was as high as 46.1 % when the culture conditions were optimal. The optimal culture condition is as follows： the final concentration of IPTG in the LB medium with 0.2% glucose was 0.5 retool/L, IPTG was added when the OD600 reached approximately 0.5 and the flask was incubated for another seven hours in the 30℃ shaking airbath. The part of dissoluble protein expressed could be increased 10%-15% when the temperature was lowered from 37℃ to 30℃. Conclusion： The culture conditions for preparing recombinant human hepatocellular carcinoma associated antigen SMP-30 have been established.