摘要
利用PCR的方法从假肠膜明串珠菌(Leuconostoc pseudomesenteroides 1159)的DNA中扩增得到D-乳酸脱氢酶基因,构建重组克隆质粒(pMD19-T-DLDH),转化DH5α进行蓝白斑筛选,得到阳性克隆提取质粒双酶切验证。连接pET-32a表达质粒,构建重组表达质粒,提取质粒双酶切、测序验证,测序结果与原序列基本相符。得到重组表达质粒pET-32-DLDH转化BL21大肠杆菌IPTG诱导表达,SDS-PAGE显示有明显大小约为19 ku的特异蛋白条带。
D-lactate dehydrogenase gene was amplified from DNA of Leuconostoc pseudomesenteroides 1 159 by PCR, recombinant cloning plasmid （pMD19-T-DLDH） was constructed and transformed into DH5ot, then blue-white screening was carried out, positive clones were obtained, plasmids were extracted and Double Digests verification was completed. Expression plasmid pET-32α was connect- ed to construct recombinant expression plasmid, plasmids were extracted, and Double Digests and sequencing verification were completed. The sequencing results were basically consistent with the o- riginal sequence. Recombinant expression plasmid pET-32-DLDH was transformed into BL21 E. coli IPTG to induce expression. SDS-PAGE showed that there was obviously a specific protein band which was about 19 ku.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2016年第5期533-537,共5页
Journal of Jilin Agricultural University
基金
基金项目:吉林省高等学校秸秆综合利用高端科技创新平台[吉高平合字(2014)C-1]
关键词
假肠膜明串珠菌
D-乳酸脱氢酶基因
克隆
表达
Leuconostoc pseudomesenteroides
D-lactate dehydrogenase gene
cloning
expression
