乳酸乳球菌Nisin抗性基因nisI的克隆及作为筛选标记的研究

Cloning of Nisin resistent gene nisI from Lactococcus lactis and its application as a food grade selection marker

摘要:

摘要:【目的】以nisI作为食品级选择标记构建大肠杆菌-乳酸乳球菌穿梭载体。【方法】根据GenBank报道的乳链菌肽Nisin抗性基因nisI的序列，以pLEB590为模板，设计特异性引物，PCR扩增出nisI片段，经TA克隆、酶切鉴定及测序之后，亚克隆至大肠杆菌-乳酸乳球菌穿梭载体pMG36e中，重组子命名为pMG36e-NisI，再将pMG36e-NisI电转化至对Nisin敏感的乳酸乳球菌MG1363中。【结果】获得的重组菌MG1363/pMG36e-NisI在含有20 IU Nisin/mL的培养基中生长情况良好，遗传性能稳定，这表明nisI基因赋予了宿主菌MG1363对Nisin的抗性。【结论】nisI可以作为筛选标记用于食品级表达载体的构建。

关键词:关键词: 乳酸乳球菌;Nisin抗性基因nisI;穿梭载体pMG36e;筛选标记

Abstract:

Abstract: [Objective] To construct an E.coli-L.lactis shuttle expression vector with nisI as a food grade selection marker. [Methods] According to the sequence of Nisin resistant gene nisI reported by GenBank, the nisI fragment was amplified by polymerase chain reaction with pLEB590 as template. After being sequenced, the amplicon was confirmed by Blast from NCBI. Then, the nisI was subcloned into the E.coli-L.lactis shuttle vector pMG36e, resulting in the plasmid pMG36e-NisI. The recombinant strain MG1363/pMG36e-NisI was obtained when the plasmid pMG36e-NisI was transformed into L.lactis MG1363 competent cell by electroporation. [Results] When the medium contained 20 IU Nisin/mL, the recombinant strain carrying pMG36e-NisI showed the same growth curve and genetic stability as L.lactis MG1363. [Conclusion] The nisI gene could be used as a selection marker for construction of a food grade expression vector.

Key words:Keywords: Lactococcus lactis; Nisin resistant gene nisI; shuttle vector pMG36e; selection marker