摘要
细菌视紫红质(bacteriorhodopsinBR)是极端嗜盐菌(Halobacteriumhalobium)紫膜上的一种光能转换色素蛋白,是一种极有前途的生物光电材料.野生型BR蛋白达不到实用光电材料的要求,因此,通过对细菌视紫红质基因(br基因)的定点改造,构建和表达BR突变体成为生物光电材料研究中的一个热点.以野生型br基因为模板,通过连续PCR的方法向br基因引入一个点突变,并将之克隆至pUC19载体中.将经测序鉴定的br突变基因重组到穿梭质粒pNov-R中并转化BR缺陷型嗜盐菌L33,得到紫色阳性克隆.经PCR分析和Western杂交检测表明,阳性克隆中构建的br突变基因获得了表达.
Bacteriorhodopsin(BR) is an integral membrane protein from Halobacterium halobium that functions as a lightdependent proton pump. It is known as a promising biological photoelectric material. Sitedirected mutagenesis was applied to modify the photoelectric characteristics of the BR protein from wild type strain,which plays an important role in study of the BR protein. A point mutation was introduced to the gene of bacteriorhodopsin (br gene) by successive PCR technique, and the mutant gene was obtained and cloned into pUC19 vector. After its sequence was determined, the mutant gene was cloned into a shuttle vector pNovR and transformed into BRdeficient halobacteria(strain L33). Purple clones were picked out from the plate and were identified as a BR mutant by PCR and Western blot experiment. PCR analysis showed clearly that the mutant gene was transformed into L33, and Westernblot detection result indicated that the protein was expressed and could bind to antiBR antibody specifically.
出处
《浙江大学学报（工学版）》
EI
CAS
CSCD
北大核心
2003年第3期350-353,共4页
Journal of Zhejiang University：Engineering Science
基金
国家自然科学基金资助项目(69981001).
