重组乳酸乳球菌表达纳豆激酶的研究
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摘要
纳豆激酶(Nattokinase，NK)是从日本传统食品纳豆中分离到的、具有高效溶栓活性的蛋白激酶，它能够很好地弥补传统溶血栓药物的缺陷，极有可能开发为新一代口服制剂用于血栓性疾病的预防和治疗。
本文对来源于纳豆芽孢杆菌的纳豆激酶原基因的克隆和表达开展了以下几个方面的研究工作：
（1）将编码纳豆激酶前肽、成熟肽的pro-nk序列克隆到乳酸乳球菌表达载体pNZ8048上，构建表达质粒pFY002，电转化Lactococcus lacti sNZ9000，得到重组菌株L.lactis NZ9000（pFY002）。纤溶活性实验证实了重组基因工程菌NZ9000（pFY002）能够在胞内表达出有活性的纳豆激酶；对诱导剂乳链菌肽(nisinA)的浓度、温度、诱导时间对重组工程菌乳酸乳球菌产酶的影响进行初步探究。最佳的条件是NisinA终浓度为7.5ng/ml，诱导温度28℃，诱导时间3.5h，工程菌L.lactisNZ9000/pFY002纳豆激酶的表达量达到最大值98.55U/L。
（2）以hisH基因作为同源重组的靶位点，构建基因整合型表达载体pFY008，利用营养缺陷型作为筛选标记，通过同源重组的方法将含有纳豆激酶原基因的表达盒PnisA-aprN整合到乳酸菌的基因组上，实现纳豆激酶在乳酸乳球菌中食品级的表达。构建食品级的基因工程菌，安全性高、稳定性好，能够表达出有活性的纳豆激酶，为新型溶血栓保健乳酸饮料的开发打下理论基础。
Nattokinase (NK) is a protein kinase extracted from Japanesetraditional food natto. It has shown strong fibrinolytic property. It couldmake up for the shortcomings of traditional thrombolytic drug very well,so it is likely to be used as a new generation of oral agents for thromboticdiseases.
To study on the expression of the nattokinase gene, the followingwork has been carried out.
Firstly, the pro-nk sequence was cloned into high level expressionvector pNZ8048 for Lactococcus. lactis NZ9000, the result expressionvector pFY002 was constructed and transformed into Lactococcus lactisNZ9000.The recombinant strain Lactococcus lactis NZ9000/ pFY002could express active nattokinase(NK) under the induction of nisin. Theoptimum conditions including nisinA concentration, temperature andinduction time for the expression of nattokinase in the engineering L.lactis were investigated. The optimum concentration of nisinA, theoptimum temperature and induction time for nattokinase were 7.5ng/ml,28℃and 3.5h respectively.The high yield reached 98.55U/L forNZ9000/pFY002.
Secondly, an integrative expression vector pFY008 was constructedwith hisH gene as target site for homologous recombination. In order to torealize nattokinase gene (aprN) food-grade expression in Lactococcuslactis, PnisA-aprN gene expression cassette was integrated into thegenome of Lactococcus lactis NZ9000 through double homologousrecombination. The result food-grade expression system recombinantLactococcus lactis with high safe and stable properties was constructed.Itcan produce the active nattokinase. The work mentioned above has laid a solid foundation for the development of a new lactic acid drink withthrombolytic activity.
本文对来源于纳豆芽孢杆菌的纳豆激酶原基因的克隆和表达开展了以下几个方面的研究工作：
（1）将编码纳豆激酶前肽、成熟肽的pro-nk序列克隆到乳酸乳球菌表达载体pNZ8048上，构建表达质粒pFY002，电转化Lactococcus lacti sNZ9000，得到重组菌株L.lactis NZ9000（pFY002）。纤溶活性实验证实了重组基因工程菌NZ9000（pFY002）能够在胞内表达出有活性的纳豆激酶；对诱导剂乳链菌肽(nisinA)的浓度、温度、诱导时间对重组工程菌乳酸乳球菌产酶的影响进行初步探究。最佳的条件是NisinA终浓度为7.5ng/ml，诱导温度28℃，诱导时间3.5h，工程菌L.lactisNZ9000/pFY002纳豆激酶的表达量达到最大值98.55U/L。
（2）以hisH基因作为同源重组的靶位点，构建基因整合型表达载体pFY008，利用营养缺陷型作为筛选标记，通过同源重组的方法将含有纳豆激酶原基因的表达盒PnisA-aprN整合到乳酸菌的基因组上，实现纳豆激酶在乳酸乳球菌中食品级的表达。构建食品级的基因工程菌，安全性高、稳定性好，能够表达出有活性的纳豆激酶，为新型溶血栓保健乳酸饮料的开发打下理论基础。
Nattokinase (NK) is a protein kinase extracted from Japanesetraditional food natto. It has shown strong fibrinolytic property. It couldmake up for the shortcomings of traditional thrombolytic drug very well,so it is likely to be used as a new generation of oral agents for thromboticdiseases.
To study on the expression of the nattokinase gene, the followingwork has been carried out.
Firstly, the pro-nk sequence was cloned into high level expressionvector pNZ8048 for Lactococcus. lactis NZ9000, the result expressionvector pFY002 was constructed and transformed into Lactococcus lactisNZ9000.The recombinant strain Lactococcus lactis NZ9000/ pFY002could express active nattokinase(NK) under the induction of nisin. Theoptimum conditions including nisinA concentration, temperature andinduction time for the expression of nattokinase in the engineering L.lactis were investigated. The optimum concentration of nisinA, theoptimum temperature and induction time for nattokinase were 7.5ng/ml,28℃and 3.5h respectively.The high yield reached 98.55U/L forNZ9000/pFY002.
Secondly, an integrative expression vector pFY008 was constructedwith hisH gene as target site for homologous recombination. In order to torealize nattokinase gene (aprN) food-grade expression in Lactococcuslactis, PnisA-aprN gene expression cassette was integrated into thegenome of Lactococcus lactis NZ9000 through double homologousrecombination. The result food-grade expression system recombinantLactococcus lactis with high safe and stable properties was constructed.Itcan produce the active nattokinase. The work mentioned above has laid a solid foundation for the development of a new lactic acid drink withthrombolytic activity.
引文
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[3]江晓，董明盛，江汉湖.一种食源性纤溶酶（纳豆激酶）酶学性质研究[J].中国酿造，2002，24（1）：23-25.
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[8]须见洋行，中岛仲佳，田谷直俊.血栓溶解酵素：ナットウキたゼの活性测定法[J]，研究报文，1993，88（6）：482-486
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