College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China.
College of Fisheries, Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education, Huazhong Agricultural University, Wuhan, 430070, China.
Fish Shellfish Immunol. 2019 Jun;89:43-51. doi: 10.1016/j.fsi.2019.03.031. Epub 2019 Mar 16.
Complement factor D (Df) is a serine protease, which can activate the alternative pathway by cleaving complement factor B, and involves in the innate defense against pathogens infection in teleost. In this study, we cloned, characterized the Df gene from blunt snout bream (Megalobrama amblycephala) (Mamdf), and examined its expression pattern and antimicrobial activity. The open reading frame (ORF) of Mamdf was 753 bp, encoding 250 amino acids with a molecular mass of 27.2 kDa. Mamdf consisted of a single serine protease trypsin superfamily domain, 3 substrate binding sites and 3 active sites, but no potential N-glycosylation site. Pairwise alignment showed that Mamdf shared the highest identity (94%) with grass carp (Ctenopharyngodon idellus). Phylogenetic analysis indicated that Mamdf and other vertebrate Df had a common ancestral origin. Mamdf structured with 4 introns and 5 exons. The Mamdf mRNA expressed relatively high at the intestine appearance stage during early development and constitutively expressed in various tissues with the highest expression in the kidney in healthy adults. After challenged with Aeromonas hydrophila, significant changes of Mamdf at both mRNA and protein levels in the kidney, spleen, liver and head-kidney were observed. The recombinant Mamdf protein showed antimicrobial activity against both gram-positive bacteria and gram-negative bacteria. The above results suggested the immune function of Mamdf, and would benefit further detailed Df function research in the immune process in teleost.
补体因子 D（Df）是一种丝氨酸蛋白酶，可通过切割补体因子 B 激活替代途径，并参与鱼类先天防御病原体感染。在本研究中，我们从团头鲂（Megalobrama amblycephala）（Mamdf）中克隆并鉴定了 Df 基因，并研究了其表达模式和抗菌活性。Mamdf 的开放阅读框（ORF）为 753bp，编码 250 个氨基酸，分子量为 27.2kDa。Mamdf 由单个丝氨酸蛋白酶胰蛋白酶超家族结构域、3 个底物结合位点和 3 个活性位点组成，但没有潜在的 N-糖基化位点。序列比对表明，Mamdf 与草鱼（Ctenopharyngodon idellus）具有最高的同源性（94%）。系统进化分析表明，Mamdf 和其他脊椎动物 Df 具有共同的祖先起源。Mamdf 由 4 个内含子和 5 个外显子组成。Mamdf 在早期发育过程中肠出现阶段的 mRNA 表达水平相对较高，在健康成鱼的各种组织中均有表达，在肾脏中的表达最高。在受到嗜水气单胞菌攻击后，肾脏、脾脏、肝脏和头肾中 Mamdf 的 mRNA 和蛋白水平均发生显著变化。重组 Mamdf 蛋白对革兰氏阳性菌和革兰氏阴性菌均具有抗菌活性。以上结果表明了 Mamdf 的免疫功能，将有助于进一步研究 Df 在鱼类免疫过程中的详细功能。
