Cloning,expression and bioactivity analysis of plasma-coagulase gene of Staphylococcus aureus

【Objective】 The study was to clone and express the coa gene of Staphylococcus aureus plasma-coagulase in Escherichia coli,and to analyse its activity of solidify blood plasma.【Method】 Coa gene was cloned from genomic DNA of S.aureus by PCR,and the PCR product was inserted into pET32a(+) to construct plasmid pET32a(+)/coa.The plasmid was then expressed in E.coli BL21(DE3)cells that induced by IPTG.The solidify blood plasma activity was evaluated by sample tube using the purified recombination protein.【Result】 Sequencing analysis results showed that the S.aureus Coa gene was composed of 2 106 bp encoding a mature polypeptide 701 amino acids;SDS-PAGE results showed that recombinant proteins were expressed in E.coli with molecular weight of 100 ku and the recombinant proteins accounted for 13% of the whole proteins.The content of the purified protein was 0.047 mg/mL,through activity analysis,the valency of staphylococcus aureus to solidified rabbit blood plasma was 0.012 mg/mL.【Conclusion】 The result showed that Coa protein(S.aureus plasma-coagulase) was well expressed in E.coli,and its valency of solidify rabbit blood plasma was 0.012 mg/mL.