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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 clone the pro-Nattokinase gene and realize its expression in E. coli, and to study its expression conditions. Methods Genomic DNA was isolated and purified from Bacillus subtilis as a template. The pre-Nattokinase gene was amplified by PCR and cloned into plasmid pUC19. The recombinant plasmid pNK was constructed and transformed into E. coli DH5α. The target gene fragment was inserted into expression vector pET30a to construct the recombinant expression plasmid pENK, which was transformed into E. coli BL21 (DE3) to study its expression conditions. Results The sequence analysis showed that the full length of the cloned fragment was 1152bp, which was 100% homologous with the reported sequence in Genbank. The target product was induced by IPTG after transformation of pENK108-BL21 (DE3). The result of SDS-PAGE showed that there was a specific band. The result of Western Blotting showed that the expressed product was indeed fusion protein. After transformed with pENK108-BL21 (DE3) in LB culture medium at 37 ℃ for 4h with 1mmol / L IPTG, the expressed product reached the maximum, accounting for 31% of total bacterial proteins. Time clotting showed that the expression of some fibrinolytic effect. Conclusions The pre-construction of pre-natto kinase gene chemically-inducible expression vector and the expression conditions of transformed bacteria were studied.