In Silico Cloning Of Component F-I-0 Gene Of Earthworm Fibrinolytic Enzymes

Earthworm Fibrinolytic Enzymes (EFE) are a group of proteins isolated from earthworms, which are capable of not only dissolve fibrin but also activate plasminogen into plasmin and as thrombolytics being used to cure the disease caused by thrombus. EFE has recently became a hot point of the molecular biology research. There are available about 21 nucleotide sequences and 28 protein sequences in GenBank, however, the gene of component F-I-0 has not reported so far.We aimed at F-I-0 component and assembled a sequence of 898 bp long, from dbEST in Genbank by in silico cloning utilizing bioinformatic software DNAman. The conclusion we can draw from it is that Open Reading Frame(ORF) is amongst 38 bp-778 bp encoding 246 amino acid residues.It is initiator located in 38bp and signal peptide from 38 bp to 100 bp which would be cut in mature peptide.F-I-0 N-terminal sequence is the same with amino acid sequence tranlated from 101 bp to 172 bp.It is termination codon lied in 778 bp and the poly A tail fllowed after 915 bp.We found that there are coincidence among in silico cloning result and F-I-0: N-terminator of mat_peptide and amino acids chromatogram; the protein is expected extracellular based on subcellular localization prediction; it contains trypsin-like serine protease domain by SMART and belong to serine proteases, trypsin family, histidine active site and serine active site were found by scanning in prosite database.The total RNA is extracted earthworm for RT-PCR with degenerate and special primers. And then the fragment from PCR was connected into pMD19-T vector to sequence after the recombinant plasmids had been identifed.But we found the recombinant plasmids is inconsistency with the result of in silico cloning. It may be unfit for primer design.At the same time,we explored the method of in silico cloning using protein sequences as query sequence..