Cloning an antimicrobial peptide gene from theoriental fly, Bactrocera dorsalis (Hendel) andexpressing in E. coli

• Main Authors: Yen-Ju Lee, 李彥儒
• Other Authors: Kuang-Hui Lu
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/bb2m88

碩士 === 國立中興大學 === 生命科學系所 === 107 === Fruit would be reduced from microbial infection when oriental fruit fly (Bactrocera dorsalis Hendel) laid eggs inside the fruit, triggering the investigation on the anti-microbial material associated with eggs. Firstly, it was confirmed that the egg washing solution contains antibacterial material according to the positive result of antibacterial ring test; subsequently, the SDS-PAGE analysis showed that it might be an antibacterial peptide (AMP) with a size of about 3 kDa. The mass spectrometry analysis further revealed that the AMP consisted of 28 amino acids, which is possibly derived from digestion of sarcocystatin-like protein. After that, based on the sequence of sarcocystatin-like protein gene, and followed a series of RT-PCR and RACE reactions; and finally, a full-length 489-bp cDNA sequence was completed and named Bdscys-A with an accession number MG231276 in GenBank. The Bdscys-A can transduce a protein consists of 124 amino acids, containing a signal peptide from Met1 to Ala26 and conserved domain of cystatin from Leu45 to Ser115. Furthermore, there are two suspected trypsin digestion sites located in Bdscys-A; therefore, it is speculated that the 28 amino acid comprised an antibacterial peptide with α helix structure was derived from Bdscys-A by trypsin digestion. The gene expression in adult fly showed that Bdscys-A is expressed in all of head, thorax and abdomen regions of both males and females, while the expression of the abdomen was higher than that of the head and thorax. Moreover, females expressed higher than males. In males, the Malpighian tubules showed the highest Bdscys-A, though its expression was very low; in females, the reproductive accessory glands were the highest in Bdscys-A expression compared to other organs. When Bdscys-A was expressed using E. coli protein expression system, the cDNA with the complete open reading frame (pET29a-NSPSA) could not be induced by IPTG to express recombinant protein; conversely, the signal peptide-deleted cDNA (pET29a-NSPSA) could successfully express a considerable amount of Bdscys-A protein. These results show that the signal peptide sequence of Bdscys-A completely blocked the protein synthesis in E. coli. Finally, the antimicrobial test results showed that the antimicrobial activity of Bdscys-A was not significant, and it is speculated that the antimicrobial effect of Bdscys-A could be enhanced after enzymatic digestion. It is expected, in the future, that the antimicrobial peptide would be stabilized through the substitution of amino acids, and could be developed into a widely used antimicrobial agent.