前纳豆激酶原基因温控型表达载体的构建及其表达条件的研究

CLONING AND EXPRESSION OF PRE-PRO-NATTOKINASE GENE

纳豆激酶(nattokinase,简称NK)是一种枯草芽孢杆菌蛋白激酶,该酶具有强烈溶栓活性。本研究从枯草芽胞杆菌中分离纯化得到基因组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,测序结果表明,所克隆片段全长1152bp,与GenBank中已报道序列同源性达100%。将目的基因片段与表达载体pBV220连接,构建重组表达质粒pBNK,转化大肠杆菌HB101。采用不同诱导时间、不同诱导温度(40℃、42℃和44℃)诱导表达,结果发现转化菌pBNK6-HB101在LB培养基中经30℃培养,42℃诱导表达7小时目的产物表达量达到最大,约占菌体总蛋白的25.35%,转化菌pBNK6-HB101在不同的培养基(LB和TB)中培养,无明显差异。

Nattokinase,a kind of subtilisin,which has strong fibrinolytic activity,is a serine protease.The pre-pro-nattokinase gene was amplified by PCR from genomic DNA of Bacillus subtilis and cloned into vector pUC19.Sequence analysis showed that the cloned gene was 1152bp and shared 100% homology with the reported sequence in GenBank.The cloned gene was constructed into expression vector pBV220.The recombinant expression vectors pBNK was then transformed into Escherichia coli HB101.The results showed that the expression amount reached the biggest level at 42℃ for 7h by comparison of different inducing time and different inducing temperature（40℃,42℃ and 44℃） indicating pBNK6-HB101,The expression of target protein reached up to 25.35% of total protein while the transformed bacteria HB101.The biggest expression level of transformed bacteria in LB medium induced for 7h was similar to that in TB medium cultured at 30℃ and induced at 42℃.