Cloning And Expression Of 34KD Fibrinolytic Enzyme Genes From B. Pseudomycoides

We mainly studied the cloning of fibrinolytic enzyme genes produced by microorganism and its expression in E.coli and Bacillus subtilis.The results are as follows:Gu screened Bacillus pseudomycoides with fibrinolytic enzyme activity from soil, then separated and purified a 34 kD protein.Edman degradation showed that 15 amino acids of N-terminal sequence were VTGTNAVGTGKGVLG.By using the program of BLAST on NCBI GenBank database,we found ten protein sequences with 100%homology.Then degenerate primers were designed based on the homology of coding sequence.We also added EcoRI and XhoI restriction site on both ends. Using genomic DNA of B.pseudomycoides as template,we proformed PCR and obtained a complete fibrinolytic enzyme gene-BpFE.The purified vector pGEX-4T-2 and PCR product of gene BpFE were digested by EcoRI and XhoI.After purifying the digested products respectively,we ligated these two kinds of DNA by T4 DNA ligase and constructed the recombinant plasmid pGEX-BpFE. Then BpFE gene was sequenced.The result indicated that this gene contained 1701 bp nucleotides and encoded 566 amino acid proteins.Compared with the highest similarity sequence-bacillolysin(Bacillus cereus H3081.97),only five amino acids were different.It also contained neutral zinc metallopeptidases,zinc-binding region signature.The recombinant plasmid of pGEX-BpFE was transformed into host bacterium of E.coli BL21.SDS-PAGE analysis was performed after being induced by IPTG and low temperature.The apparent molecular weight of the expression product was 98 kD.Both Casein-Plate method and Fibrin-Plate method showed that the intracellular protein had activity after being induced for 5 h at 26℃and 30℃in 1.0 mmol/L IPTG.Intracellular expression of most E.coli expression system leads inconvenience to production,while Bacillus subtilis is safe and secretes extracellular protein.So it is considered as a good receptor for the expression and secretion of heterologous protein.Using pUC19-5-2 as template,a fragment of 535 bp has been amplified by PCR which including promoter of the sacB gene,200 bp fragment similar to transcription stop signals,ribosome binding site and signals sequence encoded 29 amino acid.This fragment was digested with restriction enzymes and ligated with E.coli-Bacillus subtilis shuttle vector pMK4.After sequencing the inserted gene fragment correctly,we succeed in the construction of a E.coli-Bacillus subtilis shuttle vectors pSK4.At last,we cloned gene BpFE into the pSK4 for the further study in the expression of Bacillus subtilis.Based on genetic engineering method,the technology platform of fibrinolytic enzyme produced by microorganism has been tried to establish.That is a primary preparation for the further research on the function and mechanism of fibrinolytic enzyme,even its application potential in life of science and medical care.Our experiment will lay a necessary theory and practical foundation for the development of genetic engineering drugs.