乳酸乳球菌Nisin抗性基因的异源性表达和活性研究

Heterologous expression and activity research of a nisin resistance gene in Lactobacillus delbrueckii subsp bulgaricus

目的评估乳酸乳球菌Nisin抗性基因在德氏乳杆菌保加利亚亚种的表达活性,为建立食品级筛选标记做准备。方法将新鲜牛奶接种于含有400μg/ml乳酸链球菌素的M17平板上,在分离的菌株中筛选乳酸乳球菌,同时提取其基因组DNA和质粒DNA,根据已公布的Nisin抗性基因序列设计一对引物,以提取的基因组DNA和质粒DNA为模板进行PCR扩增,产物进行电泳分析。将目的基因插入到大肠杆菌乳杆菌穿梭质粒—pMG36e中,并在德式乳杆菌保加利亚亚种中观察目的基因的表达活性及其作为筛选标记的活性。结果成功筛选出对Nisin具有抗性的乳酸乳球菌,进行目的基因扩增后成功将目的基因转化到L6032中,并表达出Nisin抗性。结论在德式乳杆菌保加利亚亚种中成功克隆及表达出Nisin抗性基因。

Objective To evaluate the activity of nisin resistance gene （nsr） in transformant Lactobacillus delbrueckii subsp. bulgaricus, and prepare for establishing a food-grade selective marker. Methods Inoculated fresh milk in M17 broth containing 400μg/ml nisin and incubated at 30℃for 16 h. Then picked out the suspected colonies, and extracted genomic DNA and plasmid DNA. Based on the nsr gene sequence information, PCR amplification was performed with genomic DNA or plasmid DNA, elec-trophoretic analysis was used to analyze the products. The target gene was inserted into pMG36e, and recombinant plasmid pMG36e-nsr was transformed into Lactobacillus delbrueckii subsp. bulgaricus （L6032） competent cells. Results Six nisin resistant stains were identified as Lactococcus lactis. The target gene was cloned into E. coli-Lactobacillus shuttle vector pMG36e. The Nisin resistibility was obtained when the recombinant plasmid pMG36e-nsr was transformed into Lactobacillus delbrueckii subsp. bulgaricus L6032 competent cells. Conclusions The nsr gene was successfully cloned and expressed in Lactobacillus delbrueckii subsp. bulgaricus L6032.