Shin Hak Sup, Park Soon-Ik
a Department of Biological Sciences , Dankook University , Cheonan , Republic of Korea.
Prep Biochem Biotechnol. 2019;49(3):279-285. doi: 10.1080/10826068.2018.1541807. Epub 2019 Feb 15.
In this study, we report a novel member of the attacin family from Hermetia illucens. The cDNA clone encoding the attacin-like protein was isolated by screening a cDNA library prepared from immunized fat body. The complete 510 bp cDNA of HI-attacin was predicted to encode a protein of 169 amino acids with a molecular weight of 17.7 kDa. The putative mature protein of H. illucens attacin (HI-attacin) had 50% identity with that of Bactrocera dorsalis attacin B. Phylogenetic analysis revealed that the HI-attacin was separated from the other dipteran attacins with a high bootstrap percentage. Compared to that in the other dipteran attacins, the G1 domain of HI-attacin was shorter and the sequence of the G2 domain of HI-attacin was more conserved than that of the G1 domain. We produced the recombinant attacin (rHI-attacin) protein using a prokaryotic expression system to confirm its antibacterial character. The rHI-attacin was produced as inclusion bodies and refolded by on-column refolding. rHI-attacin exhibited antibacterial activity against both Escherichia coli and methicillin-resistant Staphylococcus aureus (MRSA). Using real-time PCR, the expression of HI-attacin was was barely detected before the immunization, but was mostly evident in the fat body after immunization.
在本研究中，我们报道了一种来自黑水虻的新型attacin家族成员。通过筛选从免疫脂肪体制备的cDNA文库，分离出编码attacin样蛋白的cDNA克隆。预测HI-attacin的完整510bp cDNA编码一个169个氨基酸的蛋白质，分子量为17.7kDa。黑水虻attacin（HI-attacin）的推定成熟蛋白与桔小实蝇attacin B的成熟蛋白具有50%的同一性。系统发育分析表明，HI-attacin以较高的自展百分比与其他双翅目attacins分离。与其他双翅目attacins相比，HI-attacin的G1结构域较短，且HI-attacin的G2结构域序列比G1结构域更保守。我们使用原核表达系统制备重组attacin（rHI-attacin）蛋白以确认其抗菌特性。rHI-attacin以包涵体形式产生并通过柱上复性进行重折叠。rHI-attacin对大肠杆菌和耐甲氧西林金黄色葡萄球菌（MRSA）均表现出抗菌活性。使用实时PCR，在免疫前几乎检测不到HI-attacin的表达，但在免疫后主要在脂肪体中明显表达。
