Gene cloning and function analysis of complement B factor-2 of Apostichopus japonicus.

摘要

In this study, a homologue of complement B factor (AjBf-2, GenBank ID: JN634069.1) was cloned and characterized from Apostichopus japonicus by using bioinformatics methods and molecular biotechnologies including homology cloning and RACE. The full-length cDNA of AjBf-2 was composed of 3261bp. The sequence shows 268bp in the 5鈥睻T region, 395bp in the 3鈥睻T region, and 2595 bp in the open reading frame. AjBf-2 gene encodes 865 amino acids. The deduced amino acids sequence and domain structure of AjBf-2 gene show significant similarity to the vertebrate Bf/C2 family protein. AjBf-2 is a mosaic protein. It has a deduced molecular mass of 96.8 kDa, with a conserved site for a D factor. AjBf-2 is composed of five short consensus repeats, a von Willebrand Factor domain, a serine protease domain and an Mg2+ binding site. It has eight consensus recognition sites for N-linked glycosylation and four cAMP- and cGMP-dependent protein kinase phosphorylation sites. Phylogenetic analysis of AjBf-2 compared with other species Bf shows that A. japonicus has a close evolutionary relationship with Strongylocentrotus purpuratus and Carcinoscorpius rotundicaud. It can be speculated that Bf in invertebrate is the ancestor of Bf in vertebrate. The result of RT-PCR shows that the AjBf-2 gene is expressed in every tested tissue of A. japonicus, and is especially high in the coelomocyte and the body wall. The expression tendency in coelomocyte and the body wall are approximately the same. After LPS induction, the expression of AjBf-2 gene peaks at 12 h in coelomocyte and 3 h in the body wall.