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前纳豆激酶原基因化学诱导型表达载体的构建及鉴定

Cloning and expression of pre-pro-nattokinase gene
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摘要 目的克隆前纳豆激酶原基因,实现其在大肠杆菌中的表达,并对其表达条件进行研究。方法从枯草芽孢杆菌中分离纯化基因组DNA作为模板,通过PCR扩增前纳豆激酶原基因,将其克隆到质粒pUC19中,构建重组克隆质粒pNK并转化大肠杆菌DH5α;将目的基因片段与表达载体pET30a连接,构建重组表达质粒pENK,转化大肠杆菌BL21(DE3),对其表达条件进行研究。结果序列检测结果表明,所克隆片段全长1152bp,与Genbank中已报道序列相比较,同源性达到100%。转化菌pENK108-BL21(DE3)经IPTG诱导表达目的产物,SDS-PAGE检测结果显示具特异条带,Western Blotting检测结果表明,表达产物确系目的融合蛋白。转化菌pENK108-BL21(DE3)在LB培养基中经37℃培养,1mmol/L IPTG诱导4h后,目的产物表达量达到最大,约占菌体总蛋白的31%。时间凝块法显示表达物具有一定的纤溶作用。结论成功构建前纳豆激酶原基因化学诱导型表达载体,并对转化菌的表达条件进行了研究。 Objective To colone the pre-pro-nattokinase gene and study the expression condition in Escherichia coli.Methods The pre-pro-nattokinase gene was amplified by PCR from genomic DNA of Bacillus subtilis and cloned into vector pUC19.The cloned gene was constructed into expression vector pET30a.The recombinant expression vectors pENK were then transformed into Escherichia coli BL21(DE3).Results Sequence analysis showed,that the cloned gene was 1152bp and shared 100% homology with the reported sequence in GenBank.Western blotting assay showed that the target fusion protein was detected when the transformed bacteria BL21(DE3)(containing pENK108) were induced by IPTG.The expression level of target protein in BL21(DE3) reached up to 31% when the bacteria were cultured at 37℃and induced by 1mmol/L IPTG for 4h in LB.Fibrinolytic activity was showed in the method of time clot when the transformed bacteria BL21(DE3)(containing pENK108) were induced by IPTG.Conclusion The pre-pro-nattokinase gene has been colone and expressed in Escherichia col.
出处 《中国国境卫生检疫杂志》 CAS 2012年第2期78-82,共5页 Chinese Journal of Frontier Health and Quarantine
基金 内蒙古自然科学基金资助(200408020318)
关键词 前纳豆激酶原基因 纳豆激酶 克隆 表达 纤溶作用 Pre-Pro-nattokinase gene Nattokinase Clone Expression Fibrinolytic activity
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参考文献13

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