家蝇溶菌酶1基因的克隆、序列分析及原核表达

Cloning,sequence analysis and prokaryotic expression of the Lysozyme 1 Gene from Musca domestica

关键词:  家蝇  家蝇溶菌酶1  克隆  序列分析  原核表达

Key words:  Musca domestica  Md-lysozyme 1  Clone  Sequence analysis  Prokaryotic expression

摘要:

采用cDNA末端快速扩增技术(RACE),对鸡致病性大肠杆菌诱导家蝇幼虫抑制性消减文库(SSH)中筛选得到家蝇溶菌酶1基因((Musca domestica lysozyme-1,MdL-1)进行扩增,测序分析得到一个全长为537bp的cDNA片段,其开放阅读框(ORF)432bp,与Genbank中登录号为AY344589.1基因同源性为96%。构建重组表达质粒pET-32a-MdL-1,转化大肠杆菌BL21(DE3),异丙基-β-D-硫代吡喃半乳糖苷(IPTG)诱导表达,十二烷基磺酸钠－聚丙烯酰胺凝胶电泳(SDS-PAGE)分析显示,表达产物大小约为34KDa,与预期蛋白大小一致。结果表明利用RACE技术克隆得到MdL-1全长基因并在大肠杆菌中获得了表达,为进一步研究该蛋白的生物学及免疫学活性奠定了基础。

Abstract:

a partial cDNA sequence of Lysozyme 1 gene from Musca domestica was obtained by SSH Library of Musca domestica larvae Upon Infection with Enteropathogenic Escherichia coli, Its full-length was amplified by cDNA end rapid amplification technology(RACE), Sequence analysis showed that the full-length cDNA of MdL-1 is 537bp, the open reading frame (ORF) is 432bp, the homologies of this gene with the MdL-1 from GenBank were up to 96%. Recombinant expressed plasmid was reconstructed with pET-32a, which in Escherichia coli BL21 (DE3) induced by IPTG and verified by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the fusion protein relative molecular weight was 34kDa. These results revealed that MdL-1 cloned by RACE can express successfully in Escherichia coli BL21(DE3) and establish the basis for further researches in biology activity especially in Enteropathogenic Escherichia coli and immunologic Activity.