培养条件对pTrxAR质粒在E.coliGI724中孕育量的影响

EFFECTS OF CULTIVATION CONDITIONS ON THE QUANTITY OF pTrxAR PROPAGATED IN E. coli GI724

硫氧还融合表达系统是Invitrogon公司近年来发展的以E .coli硫氧还蛋白作为融合伴侣的融合表达系统。其重组质粒的诱导表达需在E .coliGI系列菌株中进行。本文对载体质粒pTrxFus及其携带编码hARLBD基因的重组质粒pTrxAR在E .coliG1 72 4株中的质粒孕育量与生长培养条件的关系进行了研究。结果表明 ,菌株在平板RM培养基上的活化生长时间及菌体液体培养时的温度对其质粒孕育量影响极大 ,其次为液体培养基中Ampicillin浓度。菌体在35℃ ,1 0 0ug/mlTrp的诱导条件下 ,其重组质粒在数小时内迅速丢失。通过菌体质粒的提取、测定 ,可为优化使用该表达体系表达hARLBD片段的表达条件提供理论指导。

ThioFusion expression system, a fusion expression system using E.coli thioredoxin as a fusion partner, was developed by Invitrogen Company a few years ago. To obtain induced expression of the heterogeneous gene inserted into the vector of pTrxFus, E. coli GI strains must be used. The relations between the plasmid quantity of pTrxFus and its derivate pTrxAR which carry the gene of hAR LBD propagated in E. coli GI724 and the cultivation conditions were investigated in this study. It was shown that the time of activating cultivation of the strain on soild RM medium and the temperature of the cultivation of the bacteria in liquid medium were the most important factors affecting the plasmid quantity, followed by Ampicillin concentration in the liquid medium. Under the induction condition of 35℃ and 100 μg/mL Trp, the bacteria lost their plasmids within several hours. The method of plasmid extraction and determination can be used as a guide in optimizing expression conditions of the hAB LBD within the ThioFusion expression system.