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目的克隆点带石斑鱼hepcidin前体cDNA序列,构建其成熟肽pET-32a融合表达载体。结论根据已报道的硬骨鱼类hepcidin cDNA序列设计简并引物,以点带石斑鱼(Epinephelelus malabaricus)肝脏为材料,通过RT-PCR扩增hepcidin cDNA序列,用比对推导的氨基酸序列预测其成熟肽段。再将成熟肽cDNA序列亚克隆至pET-32a构建原核表达并进行原核融合表达。结果获得点带石斑鱼hepcidin-like抗菌肽前体cDNA序列1条,GenBank登录号为HM474788。DNA序列的Blast分析以及推导氨基酸序列NJ进化树分析表明,HM474788是点带石斑鱼第1条报道的抗菌肽hepcidin cDNA序列。成功构建了该序列的成熟肽原核融合表达载体pET-32a-hepcidin(EM1),并成功在大肠杆菌origami(DE3)中表达。结论成功克隆点带石斑鱼hepcidin-like抗菌肽前体cDNA序列,成熟肽成功在大肠杆菌中融合表达,为后续有关石斑鱼hepcidin的功能研究与实践应用奠定重要基础。
Objective To clone the cDNA sequence of the hepcidin precursor of grouper and construct its mature peptide pET-32a fusion expression vector. Conclusions Degenerate primers were designed according to reported hepcidin cDNA sequence. Epistaxis malabaricus liver was used as a material to amplify hepcidin cDNA sequence by RT-PCR. The amino acid sequence of Hepcidin was predicted to be mature Peptides. Then, the mature peptide cDNA sequence was subcloned into pET-32a for prokaryotic expression and prokaryotic expression. Results The cDNA sequence of the hepcidin-like antimicrobial peptide was obtained from GenBank with the accession number HM474788. Blast analysis of DNA sequence and deduced amino acid sequence NJ phylogenetic tree analysis showed that HM474788 was the cDNA sequence of the antibacterial peptide hepcidin reported in Article 1 of spotted grouper. The mature prokaryotic fusion expression vector pET-32a-hepcidin (EM1) was successfully constructed and successfully expressed in E. coli origami (DE3). Conclusion The cloned cDNA of hepcidin-like antimicrobial peptide was successfully cloned and the mature peptide was successfully expressed in Escherichia coli, which lays an important foundation for the functional research and practical application of hepcidin.