Nisin抗性菌株的筛选及Nisin抗性基因的克隆

doi:10.7506/spkx1002-6630-201113034

食品科学 ›› 2011, Vol. 32 ›› Issue (13): 156-160.doi: 10.7506/spkx1002-6630-201113034

Nisin抗性菌株的筛选及Nisin抗性基因的克隆

李艳芳1，曹健1,2,*，吴兴泉1，万谦1，张洁琼1，田广兰1

1.河南工业大学生物工程学院 2.中原工学院

出版日期:2011-07-15 发布日期:2011-07-02
基金资助:
河南省高校杰出科研人才创新工程资助项目(2006KYCX008)

Screening of Nisin-resistant Strain and Cloning of Its Nisin-resistant Gene

LI Yan-fang1，CAO Jian1,2,*，WU Xing-quan1，WAN Qian1，ZHANG Jie-qiong1，TIAN Guang-lan1

1. College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China； 2. Zhongyuan University of Technology, Zhengzhou 450007, China

Online:2011-07-15 Published:2011-07-02

摘要/Abstract

摘要： 利用乳酸链球菌肽(Nisin)抗性基因常与乳糖利用基因位于同一质粒上这一特性，在含300IU/mL Nisin和质量浓度为0.004g/100mL溴甲酚紫的选择性培养基上，从新鲜牛奶中初筛到5株Nisin抗性菌株。经脱脂乳培养基培养、革兰氏染色和镜检，初步确定为乳球菌。分别以5株抗性菌株的基因组和质粒DNA为模板，采用PCR对其Nisin抗性基因(NSR)保守序列进行了扩增，从其中3株抗性菌株(分别命名为N1、N2、N3)的质粒DNA上得到大小约400bp的目的基因产物。经16S rDNA鉴定，进一步确定3株抗性菌株均为乳酸乳球菌。分别以N1、N2、N3的质粒为模板，采用PCR扩增其全长NSR基因，均得到大小约1000bp的目的产物。将从N1得到的NSR基因全长产物与pMD-19T进行连接测序，结果显示，其与已发表序列的相似性达99%，可确定其为NSR基因，且位于抗性菌株N1的质粒上。

关键词: nisin抗性基因（NSR）, nisin抗性菌株, 克隆, 筛选

Abstract: According to the fact that Nisin-resistant gene and lactose-fermenting gene often have the same locus, five Nisin-resistant strains were screened from fresh milk with a selective medium (GM17) supplemented with 0.004 g/100 mL bromocresol purple and 300 IU/mL Nisin. By using skimmed culture medium, Gram s staining and microscopic examination, five strains were identified as Lactococcus lactis. PCR amplification of Nisin-resistant gene (NSR) was carried out with chromosome and plasmid DNA as templates. An expected PCR product with 400 bp was achieved from three out of five strains. The 16S rDNA sequence analysis revealed that three strains named as N1, N2 and N3 belonged to Lactococcus lactis. The full-length of Nisin-resistant determinant gene (NSR) with 1 kb in size was cloned with the plasmids N1, N2 and N3 as templates, respectively. The PCR product of N1 was sequenced and the amplicon exhibited 99% similarity with the published sequence and was therefore confirmed as NSR by Blast analysis.

Key words: Nisin-resistant strain, screening, Nisin-resistant gene (NSR), cloning

中图分类号:

Q781

李艳芳，曹健，吴兴泉，万谦，张洁琼，田广兰. Nisin抗性菌株的筛选及Nisin抗性基因的克隆[J]. 食品科学, 2011, 32(13): 156-160.

LI Yan-fang，CAO Jian,，WU Xing-quan，WAN Qian，ZHANG Jie-qiong，TIAN Guang-lan. Screening of Nisin-resistant Strain and Cloning of Its Nisin-resistant Gene[J]. FOOD SCIENCE, 2011, 32(13): 156-160.

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Nisin抗性菌株的筛选及Nisin抗性基因的克隆

Screening of Nisin-resistant Strain and Cloning of Its Nisin-resistant Gene

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