摘要
目的 :获得NF κBp5 0亚基同源结构域 (RHD)cDNA及构建酵母双杂交系统的“饵”载体 ,并检测靶基因的酵母细胞毒性及自主报告基因的活性。方法 :提取人外周血单个核细胞(PBMC)的总RNA ,以RT PCR扩增NF κBp5 0亚基RHD基因片段 ,重组入酵母双杂交载体pGBKT7中。应用乙酸锂法 ,将重组质粒转化酵母细胞AH10 9,在SD/ Trp选择培养基上培养 ,观察转化株的生长情况 ;用滤膜影印法验证靶基因有无自主报告基因的活性。结果 :经酶切及PCR鉴定 ,重组质粒中插入片段的大小与预期的结果相符。测序结果表明 ,其基因序列正确 ,无读码框移位 ,命名为pGBKT7 p5 0。转化酵母细胞后 ,对酵母细胞无毒性也无自主报告基因的活性。结论 :pGBKT7 p5 0可作为酵母双杂交系统中的“饵”载体 ,用于后续的肽库筛选 ,捕捉与靶基因相互作用的多肽。
AIM: To clone NF-κB p50 Rel homology domain (RHD) gene and construct the “bait” vector in yeast two-hybrid system, and detect the yeast cell toxicity and autonomous reporter gene activity of target gene. METHODS: Total RNA was extracted from human peripheral blood mononuclear cells and NF-κB p50 RHD gene was amplified by RT-PCR, and cloned into pGBKT7. The recombinant plasmid was transformed into yeast AH109. The growth condition of the transformants was observed in the selected medium SD/-Trp. The reporter gene activity of target gene in the yeast cells was verified by filter blotting. RESULTS: Using restriction enzyme digestion analysis and PCR, the length of inserted gene was confirmed correct. Sequencing result indicated that the sequence of the inserted gene and its open reading frame were completely correct, and then the recombinant plasmid was named pGBKT7-p50. p50 RHD gene had neither autonomous reporter gene activity nor yeast cytotoxicity. CONCLUSION: As a bait plasmid, pGBKT7-p50 could be used in yeast two-hybrid system to screen and capture the polypeptides which interact with p50 RHD.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2004年第4期419-421,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
国家重点基础研究发展规划 (973)项目资助 (No .G1 9990 542 0 3)
国家自然科学基金资助 (No .30 0 80 0 0 9)
重庆市科委基金项目资助 (No .2 0 0 0 631 9)
