论文部分内容阅读
目的制备新型免疫佐剂环二鸟苷酸(c-di-GMP)并进行纯化。方法利用前期重组构建并在大肠杆菌中成功表达的霍乱弧菌VCA0956蛋白(含有GGDEF结构域)作为合成酶,在反应体系中采用酶促反应将2分子鸟苷酸GTP催化缩合成一分子c-di-GMP。利用反相液相色谱仪分离纯化粗产物中的c-di-GMP,柱温25℃,流动相为乙腈-10 mmol/L乙酸铵溶液(体积比为5∶95逐渐变为30∶70),流速1 m L/min。结果 c-di-GMP从反应粗产物中被有效的分离纯化,同时,经质谱分析仪分析鉴定,获得高纯度的c-di-GMP。结论成功从酶促反应体系中分离并纯化了c-di-GMP。
Objective To prepare and purify c-di-GMP, a novel immune adjuvant. Methods Vibrio cholerae VCA0956 protein (containing GGDEF domain), which was previously constructed recombinantly and successfully expressed in Escherichia coli, was used as a synthase. The enzymatic reaction was used to catalyze the condensation of two molecules of guanylate GTP into one molecule of c- di-GMP. The crude product c-di-GMP was separated and purified by reversed-phase liquid chromatography. The mobile phase consisted of acetonitrile-10 mmol / L ammonium acetate solution (the volume ratio was 5:95 and gradually changed to 30:70) , Flow rate 1 m L / min. As a result, c-di-GMP was effectively isolated and purified from the crude reaction product, and at the same time, it was identified by mass spectrometry analysis to obtain high-purity c-di-GMP. Conclusion The c-di-GMP was successfully isolated and purified from the enzymatic reaction system.