摘要
从原始质粒pDsRed2-1中分离红色荧光蛋白基因DsRed2、构建原核表达载体pGEX4T1-DsRed2，进行了原核表达。结果表明：目标DNA序列含有由678bp构成的开放阅读框（ORF），编码由225个氨基酸构成的蛋白（红色荧光蛋白，简称RFP2）。与参考序列相比，目标DNA序列在其ORF内存在一个单碱基的同义突变，所编码的目的蛋白氨基酸序列不变。DsRed2基因在常用的表达菌株BL21（DE3）和克隆菌株DH5et中都能够表达出目的蛋白RFP2，使菌落呈现红色。在BL21（DE3）中，在1～5h内，随诱导时间的延长，RFP2的表达量迅速增加，此后增加不明显。在原核细胞中表达出的RFP2，大多以难溶性包涵体形式存在，仅少量溶于细胞质中，但都能正常显示出红色。RFP2为单体红色荧光蛋白，且在自然光下即可激发出红色荧光；DsRed2作为报告基因，可以非常简单、方便地区分出阳性和假阳性菌落。
In this study, cloning and prokaryotic expression of DsRed2 gene encoding red fluorescent protein （RFP2） were conducted. The results showed that DNA sequence of DsRed2 gene involved a 678 bp ORF encoding a red fluorescent protein RFP2 that comprised of 225 amino acids. The gene cloned had a single-base synonymous mutation within its ORF resulting in producing an e- qual protein. The DsRed2 gene could express in the expression strain BL21 （DE3） as well as in the cloning strain DH5tx. The quantity of target protein expressed in BL21 （DE3） cells increased with the increase of induction time from 1 to 5 h, but not obviously increased hereafter. The RFP2 could dissolve in cytoplasm, but most of the RFP2 appearing red existed in hard-soluble inclusion bodies. RFP2 was a monomeric red fluorescent protein appearing red under natural light, and DsRed2 could be used as a reporter gene to easily distinguish positive and false positive clones.
出处
《吉林农业大学学报》
CAS
CSCD
北大核心
2016年第4期420-425,共6页
Journal of Jilin Agricultural University
基金
延边大学校科研启动基金项目(2011800-950118010)
吉林省高等教育专项资金项目(012013002)
关键词
DsRed2
单体红色荧光蛋白
原核表达
包涵体
DsRed2
monomeric red fluorescent protein
prokaryotic expression
inclusion body
