【作者】 郝凤奇； 王春凤； 杨桂连； 叶丽萍； 杨中娜；

【Author】 HAO Feng-qi WANG Chun-feng YANG Gui-lian YE Lip-ing YANG Zhong-na(Animal Microecology Laboratory,College of Animal Science & Technology,Jilin Agricultural University,Changchun 130118)

【机构】 吉林农业大学动物科技学院动物微生态学研究室；

【摘要】 本研究旨在以一株分离自牛乳中乳酸乳球菌染色体 DNA 为模板,采用 PCR 技术扩增出乳链菌肽生物合成的双组分调控元件(nisRK 基因)。试剂盒回收纯化 nisRK 基因后将其克隆入以氨苄青霉素为选择压力的 pGEM-T 克隆载体中,再转化E.coli DH5α感受态细胞,筛选阳性克隆,提取重组体,进行酶切鉴定,并对 nisRK 基因进行序列测定,与已知序列进行同源性比较,结果表明成功的克隆出 nisRK 基因,全长为2035 bp,与国外报道的 nisRK 基因同源性高达99.9%.为构建"食品级" 乳链菌肽诱导表达载体系统奠定了基础.更多还原

【Abstract】 Using chromosome DNA of Lactococcus Lactis isolated from milk as template,nisRK gene was amplified by PCR with Pfu DNA polymerase,and purified by purification kit.Choosing pGEM-T cloning vector,containing ampicillin resistance gene,the PCR production of nisRK gene was cloned into it.Then the ligadon was transformed into E.coli DH5α competent cells.The recombinant plasmid containing nisRK gene was selected by cleaved with restriction endonuclease Xba I and Kpn I.The result showed that the whole nisRK gene was cloned already(2035 bp).Sequence analysis indicated that nisRK gene displayed 99.9% nucleotide homology with the published abroad.The nisRK gene proved a good foundation for the construction of food-grade nisin controlled expression system(NICE).更多还原