家蝇幼虫双翅肽MdDpt I成熟肽基因的克隆与原核表达

Cloning and prokaryotic express of diptericin I gene MdDpt I mature peptide chain in Musca domestica larvae

关键词:  家蝇  家蝇双翅肽  克隆  序列分析  原核表达

Key words:  Musca domestica  Musca domestica Diptericin  Clone  Sequence analysis  Prokaryotic expression

摘要:

对鸡沙门氏菌诱导家蝇幼虫构建的抑制性消减文库(SSH)中筛选得到的家蝇双翅肽 I(Musca domestica Diptericin I, MdDpt I)基因进行扩增及表达,以期为该基因生物活性的研究奠定基础。以沙门氏菌诱导家蝇幼虫cDNA为模板,采用cDNA末端快速扩增技术(RACE),对MdDpt I基因进行扩增,并对扩增产物进行测序和生物信息学分析,进一步去掉信号肽并构建重组表达质粒pET-28a-MdDpt I,转化大肠杆菌BL21(DE3),IPTG诱导表达,对表达产物进行SDS-PAGE电泳分析。MdDpt I基因全长为419bp,包含一个300bp的完整开放阅读框(ORF),编码99个氨基酸,其cDNA序列与Genbank中登录号为FJ794602.1的家蝇双翅肽基因同源性为95%。构建了家蝇MdDpt I基因的成熟肽原核表达质粒pET-28a-MdDpt-I,并获得成功表达,表达产物约为12ku,与预期结果一致。获得了MdDpt I基因的全长序列,构建了原核表达载体,并成功获得表达。

Abstract:

Purpose The study was done to clone and express gene of MdDpt I (Musca domestica Diptericin I)being screened from SSH library by salmonella, in order to lay the foundation for its bioactivity evaluation. Method Using cDNA end rapid amlification technology(RACE), MdDpt I gene’s full-length cDNA was amplified, then recombonant expression plasmid was reconstructed using pET-28a, transformed into E.coli BL21 (DE3)and then induced by IPTG, the recombinant protein was verified by SDS-PAGE. Result The full-length cDNA of MdDpt I gene was 419bp, in size, the open reading frame was 300bp, encoding a 99-aminoacid protein, the homology of this gene’s cDNA sequence with the MdDpt I from GenBank was up to 95%. The mature peptide recombonant expression plasmid pET-28a-MdDpt I of MdDpt I gene was constructed, and the protein of MdDpt I gene was expressed successfully with the size of 12ku, Conclusion The result showed that the recombinant plasmid pET-28a-MdDpt I was constructed correctly, which paved the way for further studies on the MdDpt I protein expression and its biological activities.