透明颤菌血红蛋白基因的克隆与表达
Cloning and expression of Vitreoscilla Hemoglobin in Esherichia coli
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摘要:目的 用野生型透明颤菌菌株中获得的血红蛋白基因, 构建透明血红蛋白(VHb)重组表达质粒, 并在大肠埃希菌(E.cloi)中实现表达.方法 将环境中筛选出的透明颤菌菌株, 直接通过聚合酶链式反应(PCR)克隆血红蛋白基因, 以质粒p Bluescript SK为载体.构建透明颤菌血红蛋白重组表达质粒p Blue V, 并转化到大肠埃希菌DH-5a中, 使用CO示差光谱、十二烷基磺酸钠-聚丙烯酰胺凝胶蛋白电泳(SDS-PAGE)电泳和蛋白印记(westernblot)进行分析.结果 PCR扩增的产物透明颤菌血红蛋白基因(vgb)在琼脂糖凝胶电泳图 0.5kb处显示1条亮带, 测序结果证实与已发表的vgb序列的同源性100%.经质粒转化的大肠埃希菌菌株在16kb处有1条特征蛋白带, 而未经质粒转化的菌株则没有.结论 成功构建透明颤菌血红蛋白重组质粒, 并在大肠埃希菌中得到表达.Abstract:Objective To construct one expression plasmid containing Vitreoscilla hemoglobin gene(vgb)-pBlurscript SK and to express vgb in E.cloi DH-5 A.Methods Vitreoscilla came from our laboratory that selected from environment.Plasmid-pBlurscript SK was used vector.VHb gene was cloned by PCR technique.Recombinant expression plasmidpBlueV was constructed and was introduced into Escherichia.coli.The expression production of VHb in the host was ver ified by means of the CO-binding difference spectra and the SDS-PAGE and Western blot.Results VHb gene molecular weight was 0.5 kb.DNA sequencing confirmed that the fragments in pBlue V was vgb gene.The homology was 100% compared with the published vgb gene fragments.Conclusion Recombinant expression plasmid containing Vitreoscilla hemoglobin gene(vgb)was successfully constructed and expressioned in E.cloi.
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