[In silico cloning of Efp-0, a novel earthworm fibrinolytic enzyme gene and verification of its coding region by RT-PCR].
There are four different types of N-terminal amino acid sequences (F-I-0, F-I, F-II, F-III) in the multicomponents of earthworm fibrinolytic enzymes (EFE). In GenBank 21 nucleic acid sequences of EFE have been reported. Among them, most of the N-terminal amino acid sequences belong to the F-III type,few belong to the F-II type. Only one is similar to the F-I type, but none to F-I-0. In this research we hoped to obtain the gene encoding component F-I-0 of EFE by the bioinformatics tools. Based on the N-terminal amino acid sequence VVGGSDTTIGQYPHQL of the F-I-0 type from Lumbricus rubellus, a nucleic acid sequence was obtained by in silico cloning from dbEST of Lumbricidae using the software DNAMAN. A new gene of EFE from Eisenia foetida was successfully obtained by RT-PCR using specific primers designed according to this sequence. The new gene named EfP-0 was cloned in pMAL-c2x and expressed as the fusion protein MBP-EfP-0 in the supernatant of lysate. The fusion protein MBP-EfP-0 purified by affinity chromatography had hydrolytic activity on casein plate. Sequencing result shows, EfP-0 has 678bp and encodes a protein of 225 amino acids. The protein is a serine protease belonging to trypsin family. It has similar amino acid composition to F-I-0. BLAST in GenBank shows that the similarity is lower than 40% between EJP-0 gene and other EFE genes. By this we conclude that EfP-0 gene of EFE is a novel gene and it is the first time to be reported, its accession number for Genbank is DQ836917.
Zhao
Xiao-Yu
XY
College of Life Sciences, Hebei University, Baoding 071002, China. uyoaix@yahoo.com.cn
Gao
Shan
S
Cui
Da-Ling
DL
Geng
Feng-Ting
FT
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D019295:Computational Biology; D030541:Databases, Genetic; D010450:Endopeptidases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D020224:Expressed Sequence Tags / Q000378:metabolism; D008969:Molecular Sequence Data; D009835:Oligochaeta / Q000201:enzymology* / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D010450:Endopeptidases