【摘要】 利用PCR技术将透明颤菌血红蛋白基因(vgb)克隆到融合表达载体pET28 a,在大肠杆菌BL21(DE3)表达。重组蛋白在30℃诱导获得可溶表达。利用N i2+亲和层析对重组蛋白进行了纯化,得到重组的透明颤菌血红蛋白,蛋白呈红色。实验现象显示重组蛋白与血红素相结合。紫外光区和可见光区波长扫描分析显示:蛋白粗提物和纯化后的血红蛋白在230 nm和413 nm都有强吸收峰,初步表明,尽管重组蛋白比天然蛋白多36个氨基酸,重组蛋白仍然具有生理活性。诱导后4 h和6 h的培养液氨基乙酰丙酸含量分别达到17 mg/L和21 mg/L,说明重组蛋白的表达明显促进了体内hem e合成途径。更多还原

【Abstract】 In this study, the vgb gene encoding vitreoscilla hemoglobin was cloned from the genomic DNA of vitreoscilla C1 strains by PCR. The sequence cloned was accordance with that reported in Genebank（L21670）. The vgb gene could be expressed effectively after inserted into downstream of T7 promoter in pET28a. The soluble expression was obtained when induced by IPTG at 30℃.The soluble VHB is about 10% of total expression product of BL-21（DE3） at IPTG concertration 1.0 mmol/L and 2.5 mmol/L,respectively.The VHB purfied by Ni²⁺ chelating sepharose column. No other protein except VHB was seen in SDS-PAFE gel stained by Coomassive brilliant blue.Wave length scan of purified VHB was conducted,which showed there was apparently obsorance at 420 nm.The structure difference of purified VHB was determinated result which indicated two structures in the purified VHB-monomeric and dimeric VHB更多还原