摘要
细菌视紫红质 ( Bacteriorhodopsin,BR)是嗜盐菌细胞膜上唯一的一种光能转换色素蛋白 ,BR以视黄醛 ( retinal)作辅基 ,在光诱导下吸收光子后会产生一系列的光循环中间体 ,最后又回到原始状态 .由于 BR这种独特的分子结构和光循环过程 ,及其作为一种具有光敏特性的蛋白质生物分子 ,可通过生物学技术进行改造 ,正引起光子学研究及生物学研究领域的高度注意 .从 Genbank查询 br基因的全序列 ,然后采用聚合酶链反应 ( Polymerase chain reaction,PCR)技术 ,采用 DNA star软件辅助设计了上下游引物 ,以嗜盐菌 ( H alobactrium H alobium)的基因组 DNA为模板克隆获得了完整的细菌视紫红质基因 br,并且将经测序鉴定的 br基因重组到原核高效表达载体 p GEX- 4 T- 2中 ,转化大肠杆菌 E.coli.,将阳性转化子经 IPTG诱导表达获得 BR与 GST(谷胱苷肽转移酶 )的融合蛋白后 ,提取蛋白进行 SDS-PAGE电泳、Western印迹反应 ,检测表达结果为阳性 ,且表达蛋白的大小与预想的一致 .
Bacteriorhodopsin is the best understood ion transport protein and has become a paradigm for membrane proteins in general and transporters in particular. Bacteriorhodopsin is the simplest known light energy\|converting systems. It is a small protein and requires only light and a lipid bilayer to generate an electrochemical ion gradient. The bacteriorhodopsin gene was obtained from the genomic DNA of Halobactrium Halobium through PCR techniques, and cloned into the pGEMT vector. After its sequence was determined, it was then subcloned into the pGEX\|4T\|2 expression vector, and expressed in \%E.coli.\% JM109 after transformation and IPTG induction. The constructed plasmid containing GST\|br fused gene gave a high level of expression of the fused gene. Western blot analysis indicated that the fusion protein could specifically bind to anti\|GST antibody. SDS\|PAGE analysis showed that the weight of the fusion protein is correct.
出处
《浙江大学学报（工学版）》
EI
CAS
CSCD
北大核心
2001年第3期324-327,共4页
Journal of Zhejiang University：Engineering Science
基金
国家自然科学基金资助项目 (6 99810 0 1)
