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

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 domesticaDiptericin I,MdDptI)基因进行克隆、表达,并对表达产物的抑菌活性进行初步研究。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MdDptI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-MdDptI,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。借助亲和纯化获得目的蛋白,并验证目的蛋白的生物活性。MdDptI基因全长为419 bp,包含一个300 bp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MdDptI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12 ku,与预期结果一致。纯化后的目的蛋白表现出一定的抑菌活性。本试验获得了MdDptI基因的全长序列,构建了原核表达载体,并成功获得表达纯化。

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. 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. With the aid of affinity to obtain purified fusion protein, the fusion protein was verified. The full-length cDNA of MdDpt I gene was 419 bp, in size, the open reading frame was 300 bp, 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 12 ku, The purified fusion protein showed certain antibacterial activity. 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.