纳豆激酶基因重组质粒在大肠杆菌与毕赤酵母中的稳定性

Stability of Nattokinase Gene in E. coli and Yeast Pichia patoria

利用PCR方法以纳豆杆菌染色体DNA为模板扩增纳豆激酶基因片段 ,分别克隆到原核表达载体 pBV2 2 0与真核表达载体pPICZαA上 ,转化大肠杆菌HB1 0 1与毕赤酵母GS1 1 5 ,筛选重组子 ,通过限制性内切酶、PCR分析及序列测定 ,确定该重组子所携外源基因为纳豆激酶基因 ,对重组大肠杆菌与毕赤酵母中外源基因的稳定性进行研究比较 ,结果表明外源基因的插入对宿主菌的生长没有太大影响 ,重组质粒在E .coliHB1 0 1中具有良好的分离稳定性 ,而结构稳定性较差 ,而外源基因在毕赤酵母GS1 1

Nattokinase (NK) gene was amplified by PCR using bacillus subtilis chromosomal DNA as template and cloned into vector pBV220 and pPICZαA respectively. The recombinant plasmids were transformed into E. coli HB101 and yeast Pichia pastoria GS115 respectively and recombinant strains were screened. The heterogeneous NK gene was confirmed by restriction enzyme analysis,PCR and sequencing. The stability of NK gene in recombinant E. coli and yeast Pichia was compared and the results showed that transformation of NK gene had little effect on the growth of host cells. The results showed that the recombinant prokaroyotic plasmid had good segregational stability but bad structural stability while the recombinant eucrotic vector had excellent stability.