| 摘要: |
| 【目的】克隆金黄色葡萄球菌血浆凝固酶(Coa)基因，并在大肠杆菌中进行融合表达，分析重组蛋白的生物活性。【方法】用PCR法扩增金黄色葡萄球菌Coa基因，构建Coa基因原核表达载体pET32a（+）/coa，在大肠杆菌BL21（DE3）中进行诱导表达，采用SDS-PAGE法分析重组蛋白的表达水平；将重组蛋白纯化后，测定其血浆凝固效价。【结果】扩增出了2 106 bp的Coa基因，其包含1个完整的开放阅读框，编码含701个氨基酸的成熟多肽；IPTG诱导后该基因表达的融合蛋白分子质量为100 ku，目的蛋白产量占菌体总蛋白的13%；纯化的重组蛋白含量为0.047 mg/mL，其对兔血浆凝固效价为0.012 mg/mL。【结论】成功地克隆、表达了金黄色葡萄球菌Coa基因，Coa凝固血浆效价为0.012 mg/mL。 |
| 关键词: 金黄色葡萄球菌 血浆凝固酶 克隆 表达 活性分析 |
| DOI： |
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| 基金项目:山东省中青年科学家基金项目(BS2009NY002)；山东省农业重大应用技术创新课题(2009) |
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| Cloning,expression and bioactivity analysis of plasma coagulase gene of Staphylococcus aureus |
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ZHANG Hongbo1,2, YANG Hongjun3, GE Lijiang,et al4
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1.Research Centre of Dairy Cow Shandong Academy of Agricultural Science;2.College of Veterinary Science Shandong Agricultural University;3.Research Centre of Dairy Cow ,Shandong Academy of Agricultural Science;4.College of Veterinary Science ,Shandong Agricultural University
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| Abstract: |
| 【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. |
| Key words: Staphylococcus aureus plasma-coagulase cloning expression activity analysis |
