前纳豆激酶原基因化学诱导型表达载体的构建及鉴定

    郭文秀，孙志，云华，陈宇飞

    内蒙古国际旅行卫生保健中心，内蒙古 呼和浩特 010020

    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1 152bp，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，WesternBlotting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4 h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。关键词：前纳豆激酶原基因；纳豆激酶；克隆；表达；纤溶作用

    中图分类号：R115　文献标识码：B

    Cloning and expression of pre-pro-nattokinasegene

    GUO Wen-xiu, SUN Zhi, YUN Hua, CHENYu-fei

    Inner Mongolia International Travel HealthCare Center, Huhot, Inner Mongolia  010020,China

    Abstract:   Objective  To colone the pre-pro- nattokinase gene and study the expressioncondition in Escherichia coli.  Methods   Thepre-pro-nattokinase gene was amplified by PCR from genomic DNA ofBacillus subtilis and cloned into vector pUC19. The cloned gene wasconstructed into expression vector pET30a. The recombinantexpression vectors pENK were then transformed into Escherichia coliBL21 (DE3).  Results   Sequence analysis showed ,that the cloned gene was 1152bp and shared 100% homology with thereported sequence in GenBank. Western blotting assay showed thatthe target fusion protein was detected when the transformedbacteria BL21(DE3)(containing pENK108) were induced by IPTG. Theexpression level of target protein in BL21 (DE3) reached up to 31%when the bacteria were cultured at 37℃and induced by 1mmol/L IPTGfor 4h in LB. Fibrinolytic activity was showed in the method oftime clot when the transformed bacteria BL21(DE3)(containingpENK108) were induced by IPTG.  Conclusion   Thepre-pro-nattokinase gene has been colone and expressed inEscherichia col.

    Key words:   Pre-Pro-nattokinasegene; Nattokinase; Clone; Expression; Fibrinolytic activity

    《中国国境卫生检疫杂志》