表达vgb的糖多孢红霉菌载体构建与活性鉴定

Construction and activity identify on Sac.erythraea vgb expression vector

目的为了在糖多孢红霉菌(Sac.erythraea)中表达透明颤菌(Vitreoscilla)血红蛋白基因(vgb),发挥其在贫氧环境下与氧结合形成氧合态,而改变限氧时细胞原有的代谢方式,将vgb克隆于糖多孢红霉菌表达载体中。方法利用PCR技术克隆vgb,利用基因重组技术构建含有vgb的重组糖多孢红霉菌表达质粒,电穿孔法将vgb转化置糖多孢红霉菌中,鉴定采用SDS-PAGE电泳。vgb在重组糖多孢红霉菌表达产物的生物活性检测用Western blotting分析表示。结果克隆了含有vgb的重组糖多孢红霉菌表达质粒(pBlueV),分子量6.033 kb,筛选了重组糖多孢红霉菌株,重组菌株表达的血红蛋白能与1∶300的VHb抗体呈显色反应。结论vgb在糖多孢红霉菌中获得了表达,这对继续研究生产红霉素的工程菌改造,解决工业发酵工程菌高密度培养具有良好的应用前景。

Objectives To study expression vgb in Sac. erythraca, as it could combine with O2 and improve the cell growth and the protein synthesis in the hypoxic habitats. Vgb was cloned into Sac. erythraca expression vector. Methods Vitreoscilla genomic DNA as a template, Vgb was cloned by Polymerase chain reaction-PCR technique. Recombinant Sac. erythraca expression vector was structured by gene recombinant technology. Vgb was integrated into chromosomal DNA of Sac. erythraca by electrotransformation. Analysis was tested by SDS-PAGE and Western blotting. Results This research work had cloned plasmid-pBlueV, MW was 6. 033 kb, and had screened recombinant Sac. erythraca strain. Expression product of this strain could reveal colour when it was combined with anti-VHb antibody（1：300）. Conclusion Vgb was successly expressed in recombinant Sac. erythraca, it will benefit for the highter density cultivations of industrial production fermentation microorganism.