家蝇抗菌肽Cecropin-His_6融合基因的克隆、序列分析及其表达载体构建

Molecular Cloning and Sequence Analysis of Cecropin-His_6 Fusion Genefrom Musca domestica and Construction of Its Expression Vector

克隆家蝇幼虫抗菌肽Cecropin -His 6融合基因 ,构建其真核表达载体 ,为更深入的抗菌肽活性研究及制备新型肽类抗生素创造条件。该文从家蝇幼虫组织中提取总RNA ,RT -PCR扩增编码Cecropin开放阅读框cDNA序列 ,将其克隆至pUCm -T载体进行序列测定 ;然后用含 6×His标签序列的下游引物克隆Cecropin -His 6融合基因 ,并将其构建于真核表达载体pcDNA3.1(+)中 ;同时应用生物信息学对融合基因的理化性质和二级结构进行预测分析。结果表明 ,RT -PCR扩增得到约 2 30bp的目的cDNA片段 ,与Genebank中报道的家蝇CecropincDNA序列存在一个无义变异差异 (Lys:AAG→AAA) ;6×His标签成功融合到Cecropin的C端。Cecropin -His 6融合基因的克隆和真核表达载体的成功构建 ,为进一步制备抗耐药菌的肽类抗生素奠定了基础。

To clone the fusion gene of Cecropin-His6 from Musca domestica larva and construct its eukaryon expression vector.for the purpose of providing evidence for further studies of the activities of antibacterial peptides and the preparation of new type of peptide antibiotics.The total RNA,extracted from Musca domestica larva,was amplified into the sequence of cDNA encoding the ORF of Cecropin by RT-PCR,and the target fragment was further sequenced after being cloned into pUCm-T vector plasmid.the fusion gene of Cecropin-His6 was amplified by using the reverse primer with the sequence of 6×His tag and cloned into the eukaryon expression vector pcDNA3.1(+).Meanwhile,Bioinformatics analyzed and predicted the physical-chemical characteristics and secondary structure of the fusion gene.the results showed a 228bp length cDNA fragment was abtained by RT-PCR amplification,and it had 1 nucleotide variation of glycine to alanine(Lys:AAG→AAA)compared with the sequence of Musca domestica Cecropin reported in Genbank.6×His tag was fused to the C terminal of Cecropin successfully.The cloning of fusion gene encoding Cecropin-His6 and construction of its eukaryon expression vector will lay the foundation for the preparation of the peptide antibiotics.