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首页> 外文期刊>The protein journal >Molecular cloning and expression of α-Globin and β-Globin genes from crocodile (crocodylus siamensis)
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Molecular cloning and expression of α-Globin and β-Globin genes from crocodile (crocodylus siamensis)

机译：鳄鱼（crocodylus siamensis）的α-球蛋白和β-球蛋白基因的分子克隆和表达

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摘要

The first report of complete nucleotide sequences for α- and β-globin chains from the Siamese hemoglobin (Crocodylus siamensis) is given in this study. The cDNAs encoding α- and β-globins were cloned by RT-PCR using the degenerate primers and by the rapid amplification of cDNA ends method. The full-length α-globin cDNA contains an open reading frame of 423 nucleotides encoding 141 amino acid residues, whereas the β-globin cDNA contains an open reading frame of 438 nucleotides encoding 146 amino acid residues. The authenticity of both α- and β-globin cDNA clones were also confirmed by the heterologous expression in Escherichia coli (E. coli). This is the first time that the recombinant C. siamensis globins were produced in prokaryotic system. Additionally, the heme group was inserted into the recombinant proteins and purified heme-bound proteins were performed by affinity chromatography using Co2+-charged Talon resins. The heme-bound proteins appeared to have a maximum absorbance at 415 nm, indicated that the recombinant proteins bound to oxygen and formed active oxyhemoglobin (HbO 2). The results indicated that recombinant C. siamensis globins were successfully expressed in prokaryotic system and possessed an activity as ligand binding protein.
机译：本研究首次报道了暹罗血红蛋白（Crocodylus siamensis）的α-和β-珠蛋白链的完整核苷酸序列。使用简并引物通过RT-PCR克隆cDNA，并通过cDNA末端快速扩增方法克隆编码α和β珠蛋白的cDNA。全长α-珠蛋白cDNA包含423个核苷酸的开放阅读框，编码141个氨基酸残基，而β-珠蛋白cDNA包含438个核苷酸的开放阅读框，编码146个氨基酸残基。通过在大肠杆菌（E. coli）中的异源表达，也证实了α-珠蛋白和β-珠蛋白cDNA克隆的真实性。这是在原核系统中首次产生重组的唾液梭状芽胞杆菌球蛋白。另外，将血红素基团插入重组蛋白中，并使用加入了Co2 +的Talon树脂通过亲和层析进行纯化的血红素结合蛋白。血红素结合蛋白似乎在415 nm处具有最大吸光度，表明重组蛋白与氧结合并形成了活性氧合血红蛋白（HbO 2）。结果表明，重组C.siamensis球蛋白在原核系统中成功表达，并具有作为配体结合蛋白的活性。

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