Cloning, Expression And Functional Analysis Of Ovochymase Gene From Amphioxus Branchiostoma Belcheri Tsingtauense

Cloning,expression and functional analysis of ovochymase gene(OVCH) from amphioxus B.belcheri were reported in this study.A cDNA of B.belcheri OVCH(BbOvc),obtained via the method of PCR and RACE,was 1,792 bp.Its longest ORF was 1,248 bp encoding 415 amino acids with a predicted molecular mass of approximate 44.4 kDa.BbOvc had an N-terminal signal peptide.Like vertebrate ovochymase,BbOvc contained two domains,CUB:(?)1r/C1s, sea urchin protein uEGF,bone morphogenic protein and Tryp_SPc:Trypsin-like serine protease.And most catalytic residues of this protein were found in C-terminal domain.Genes encoding ovochymase were mainly found in vertebrate animals,such as human,mouse,bull,chicken,dog,toad and zebra fish,all of which could be divided into two families:ovochymase 1 and ovochymase 2.The domains of ovochymase in different species were consistent,mainly including CUB superfamily domain and Tryp_SPc superfamily domain.However,in invertebrate animals,the evolution and function of ovochymase gene have been rarely researched.The objects of this study were to isolate BbOvc gene,to examine its characteristics and expression pattern,and to determine if it is functionally similar to vertebrate ovochymase.Results of northern blotting revealed the presence of a single band of about 1.8 kb transcript in B.belcheri.In situ hybridization histochemistry and real-time PCR demonstrated that BbOvc transcript was most abundant expressed in ovary of B. belcheri.In addition,the presence of BbOvc in ovary,also examined by western blotting,suggested that the peptide synthesized in the reproductive system.Whole mount in situ hybridization analysis revealed that positive hybridization signals were present in all blastomeres of the embryos from 1-cell to morula stages,while,in gastrula and neural plate stage embryos,BbOvc transcripts were disappeared. An expression vector including the cDNA encoding for BbOvc and 5' additional tags of pET32a was constructed and transformed into E.coli cells.The recombinant peptide was induced by IPTG and purified by affinity chromatography on a Ni-NTA resin column.The recombinant ovochymase expressed in vitro shows a trypsin-like activity capable of hydrolysing the trypsin prototypic substrate N~α-benzoyl-L-arginine ethyl ester(60 U BAEE/mg),which can be inhibited by the trypsin-specific inhibitor soybean trypsin inhibitor.It also exhibits an antibacterial activity capable of inhibiting the growth of Gram-negative bacteria like Escherichia coli.Taken together,these data indicate that BbOvc is a novel ovochymase with an antibacterial activity and offer first clues to its role as an immune-relevant molecule which may protect the early embryos from pathogenic attacks.