摘要
采用重组DNA技术成功构建了Rb基因表达质粒。Rb基因片段来源于Rb cDNA克隆P2R4.7的BglⅡ844bp片段,表达质粒载体为PWR-13,受体菌是E·Coli TAP130。Rb蛋白表达量占细菌总蛋白量的5.04%。利用Rb基因表达产物制备的抗Rb多克隆克体,在视网膜母细胞瘤发病机理的研究中初步应用,获得了重要的实验结果。
In order to further investigate the structure and biological function of Rb protein and to prepare the antibody against Rb protein we obtained 844 bp BglⅡfragment from Rb cDNA clone and inserted it into PWR-13 vector to construct a Rb gene expression plasmid When the Rb Bgl Ⅱfragment was fused in-frame into PWR-13 it was operated by Lac Z promter of PWR-13 and produced a fused protein which consisted of expressed Rb protein and a small peptide from Lac Z. The Rb gone recombinant was transformed into E coli. TAP 130 by the CaCl_2 method and the transformers were cultured with inducement of IPTG. The total cellular proteins were analysed by SDSPAGE and then stained by commassie blue. We found that there was a unique band, compared with the control, at Dalton 28000. The same protein band gave a strong signal after western transfer and reaction with ^(135)Ⅰ labeled anti-Rb protein antibody made from synthetic Rb peptide. It composed more than 5%of the total bacterial proteins. We cut this band and immunized three rabbits with the pro tein following a standard protocol. Three months later the rabbits produced high titres of antibodies that reacted with both Rb gene expressive product and synthetic Rb peptide. The two kinds of antibodies made from either Rb gene expressive product or synthetic Rb peptide immunoprecipitated the same protein of about 105kd in both normal retinal cells and other tissue cells.
出处
《遗传与疾病》
CAS
CSCD
北大核心
1990年第4期193-195,共3页
关键词
RB基因
表达质粒
视网膜瘤
Rb gene Expression plasmid Retinob-lastoma
