目的 通过对影响表达SRH融合蛋白工程菌发酵的主要因素的研究,建立适宜SRH融合蛋白工程菌的发酵工艺.方法 通过对发酵过程中的pH、溶氧调控、诱导温度、诱导时机及诱导时间进行优化建立SRH蛋白的发酵工艺,利用SDS-PAGE电泳分析表达情况.进一步利用离子交换和凝胶过滤层析对目的蛋白进行纯化,并对纯化后目的蛋白的溶栓活性和抗凝活性进行检测.结果 优化后发酵条件为:发酵pH为7.0、溶氧为45％、菌体浓度达到OD600=8时开始诱导,诱导温度为41℃,诱导时间为4.5h.该发酵条件下得菌量为25g/L.SRH蛋白表达量为45％,并且90％以上为可溶性表达,纯化后蛋白纯度达95％以上,纯品SRH蛋白溶栓活性为1.80×104AU/mg,抗凝活性为100AU/mg.结论 建立了SRH融合蛋白工程菌稳定的发酵工艺,该条件下SRH工程菌发酵蛋白表达量较高,多为可溶性表达,且杂蛋白较少,发酵产物具有生物学活性.%Objective To establish the suitable fermentation process of engineering bacteria for expression of recombinant SRH by optimizing the main influence factors. Methods The optimum conditions of fermentation techniques were determined by optimizing the pH value, oxygen dissolution, OD value, and induction temperature and time. The expression of recombinant protein SRH was analyzed by SDS - PAGE, and SRH was purified through anion - exchange and gel filtration chromatography. The specific activity of SRH was measured by the fibrin plate assay and fibrin clot assay. Results The optimum conditions of fermentation techniques were;pH 7.0, oxygen dissolution 45% , OD600 =8, fermentation temperature 41℃, fermentation time 4.5h, and the amount of bacteria could reach 25g/L in this conditions. The expression level of SRH was about 45% and over 90% was expressed in the supernatant. After purification, the purity of SRH was over 95% . The fibrin plate assay indicated that the specific activity of SRH was 1. 80 x 10 AU/mg,and the fibrin clot assay indicated that the anti - thrombin activity of SRH was l00AU/mg. Conclusion In this study we established a stable and suitable fermentation technology of engineering bacteria expressing SRH. In this condition, the expression level of protein was high, the other proteins were less and the fermentation produce had biological activity.
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