摘要
目的:克隆、表达非洲绿猴层黏连蛋白受体(LR),对该受体蛋白进行纯化、复性研究。方法:采用RT-PCR从Vero-E6细胞总RNA中扩增非洲绿猴LR的cDNA序列,克隆到pGEM-TEasy载体上;将LR编码区插入到表达载体pET22b(+),转化大肠杆菌BL21(DE3);用IPTG进行诱导表达;用镍亲和柱进行亲和纯化;采用分部透析方法进行复性。结果:非洲绿猴LR基因序列与人LR的基因编码序列有16个碱基差异,但蛋白序列只有1个氨基酸的改变。LR蛋白以包涵体形式表达。采用分部透析方法可使部分蛋白得到复性。结论:克隆了首个非洲绿猴的相对分子质量为37000的LR的基因,与人LR基因序列有16个碱基差异,但其中15处都为同义突变,所以二者的蛋白质序列只有1个氨基酸改变,即293位D→E,提示LR在进化中具有很大的保守性。
Objective： The cDNA of African green monkey＇s laminin receptor（LR） was amplified and the recombinant LR was expressed and purificated. Methods： The cDNA of African green monkey＇s LR was amplified from total RNA of Vero-E6 cells and was ligated into pGEM-T Easy plasmid. Recombinant LR was expressed by using pET E.coli expression system. Then it was purified by Ni-NTA agarose chromatography and was renatured by dislysis. Results： By sequenc- ing, there were 16 nucleotides but only one amino acid differences between the green monkey and human LR. Recombinant LR was expressed in the form of inclusion body and was renatured by dislysis. Conclusion： The first 37 kD African green monkey＇s LR is amplified, there＇re 16 nucleotides with 15 samesense mutations but only one amino acid differences in 293 of＇D→E between the green monkey and human LR. It cue us there are high conservation of LR in the evolution.
出处
《生物技术通讯》
CAS
2006年第4期500-502,共3页
Letters in Biotechnology
基金
国家科技攻关课题(2004AA002100)
关键词
非洲绿猴
层黏连蛋白受体
基因克隆
蛋白表达
纯化
Cercopithecus aethiops
laminin receptor
gene cloning
protein expression
purification
