酵母提取物诱导重组大肠杆菌合成HrpNEcc蛋白的研究

 【目的】HrpNEcc蛋白是一种起细胞信号作用的蛋白激发子,通过激活植物遗传系统的多基因表达调控,诱导植物的广谱抗病性、驱虫性和抗逆性,促进植物生长发育,因此在农业生产中具有重要意义。在hrpNEcc重组大肠杆菌高密度发酵廉价诱导剂的筛选工作中,偶然发现不添加任何外源诱导剂的TB培养基对照处理也有HrpNEcc蛋白合成。本研究旨在找出引起对照处理中HrpNEcc蛋白合成的诱导因子,并研究诱导因子的来源和含量对HrpNEcc蛋白合成的影响。【方法】摇瓶发酵培养重组大肠杆菌,离心收集菌体,用考马斯亮蓝染色法测定菌体总蛋白,SDS-PAGE检测目的蛋白条带,并结合Bandscan软件计算出HrpNEcc蛋白含量。【结果】在不添加任何外源诱导剂的情况下,用TB培养基发酵生产重组大肠杆菌E. coli BL21(DE3)/pET30a(+)hrpNEcc,HrpNEcc蛋白最高产量可达301.45 mg?L-1,比在IPTG诱导下,用LB培养基发酵生产的HrpNEcc蛋白产量提高72.43%。进一步研究证实,TB培养基中的酵母提取物（yeast extract）含有某些诱导因子能够诱导hrpNEcc基因表达合成HrpNEcc蛋白,并且hrpNEcc的表达水平随酵母提取物来源和浓度的不同而存在较大差异。【结论】在不添加任何外源诱导剂的情况下,高含量的酵母提取物诱导重组大肠杆菌hrpNEcc表达合成HrpNEcc蛋白,但其诱导机制还有待进一步研究。

Study on Synthesis of HrpNEcc Protein in Recombinant Escherichia coli Induced by Yeast Extract

Objective] HrpNEcc protein is a kind of cellular signal transduction elicitor, which can induce plants acquiring broad-spectrum disease resistance, worms disperse ability, stress resistance and stimulate plants growth and development through activating multiple-gene expression and regulation of plants heredity system. During the experiment of screening for cheap inducer in high cell density fermentation of recombinant Escherichia coil, the TB medium without adding any exogenous inducer was used as a negative control, surprisingly the negative control showed high expression of HrpNEcc protein. The aim of this study was to find the factors inducing synthesis of the HrpNEcc protein, and study the influence of the sources and concentration of the factors on synthesis of the HrpNEcc protein. Method] The recombinant E. coli was cultured by flask fermentation, and cell pellets was harvested by centrifugation. Total protein was determined by the method of Coomassie brilliant blue, using bovine serum albumin as the standard. The qualitative analysis of protein was performed by SDS-PAGE, the Coomassie-stained protein bands were scanned by Bandscan software to estimate the percentage of HrpNEcc protein in total cellular proteins. And then the concentration of HrpNEcc protein was calculated. Result] The highest yield of HrpNEcc protein of the recombinant E.coli BL21(DE3)/pET30a(+)hrpNEcc reached about 301.45 mg·L~(-1), when the recombinant E. coli was cultured in TB liquid medium without adding any exogenous inducer. The yield of HrpNEcc protein is 72.43% higher than that of the recombinant E. coli which was cultured in LB liquid medium and was induced with IPTG Further studies indicated that high content of yeast extract in TB medium was responsible for the high-level synthesis of HrpNEcc protein, probably there are some inducing factors in yeast extract. And it also was found that the expression levels of hrpNEcc gene varied greatly with the concentrations and sources of the yeast extract used in the media. Conclusion] High content of yeast extract is found to be responsible for high-level synthesis of HrpNEcc protein without any exogenous inducer, but the inducing mechanism needs to be further studied.