纳豆激酶基因的克隆及其在毕赤酵母中的表达

Cloning of nattokinase gene and expression in Pichia pastoris

纳豆激酶纳是从日本传统食品纳豆中发现的一类具有溶栓效果的蛋白酶,由于其具有安全,高效,作用时间长,易吸收,廉价等优点,现在正成为一个开发治疗血栓类疾病药物的研究热点。从本实验室保存的一株高溶栓的纳豆杆菌N07出发,提取总基因组DNA,利用PCR手段扩增获得了纳豆激酶长为825bp的成熟肽基因片段。构建重组表达质粒pPICZaA-NK,经EcoR I、Xba I双酶切、PCR、测序验证得出重组表达质粒上的外源基因即为825bp的目的片段;将重组质粒pPICZaA-NK用内切酶Sac I线性化后电击导入毕赤酵母X33,通过含Zeocin的YPDS平板筛选获得重组酵母。重组酵母在BMMY培养基中发酵培养,用1%甲醇诱导目的蛋白表达。用纤维蛋白平板法检测发现发酵上清具有纤溶活性,经硫酸铵盐析、透析、Sephadex-G50过柱等步骤分离得到纳豆激酶蛋白,进行SDS-PAGE鉴定表明,表达的纳豆激酶蛋白分子量为27KD。以尿激酶为标准,实验所得纳豆激酶发酵上清液溶栓活性约为195U/mL。成功的将纳豆激酶成熟肽基因在毕赤酵母X33中表达,为纳豆激酶基因工程进一步研究奠定基础。

Nattokinase（NK） is a fibrinolytic enzyme extracted from natto a traditional food in Japan.Now it becomes a hot study for its advantages such as safety,super ability of dissolving thrombus,long half time,being absorbed easily by body and cost low.In this research,genome DNA of Bacillus natto N07 was extracted.The mature peptide gene amplified by PCR from the total DNA was 825bp.This NK gene was cloned on expression vector pPICZaA.After being analyzed by restriction enzyme EcoRI and XbaI,PCR and sequencing,it was confirmed that exogenous subcloned into vector was NK gene.The recombinant vector pPICZaA-NK was linearized and transformed into host cell Picchia pastoris x-33.Recombinant Picchia pastoris x-33 was isolated on YPDS plates containing Zeocin and it was induced expression by 1% concentration of methanol in BMMY.Fibrinogenolysis test showed thrombolytic activity.Purifide by the ammonium sulfate,dialyze,SephadexG-50 Chromatography and so on,the pure enzyme showed a single protein band in the SDS-PAGE and it is 27KD.The fibrinogenoulysis of NK purified from X33 is 195u/mL.This research has cloned and expressed the NK gene in Picchia pastoris x-33 successfully,it would be helpful in producing nattokinase by engineering.