分泌型人klotho基因酵母双杂交诱饵载体的构建及其毒性.docVIP

分泌型人Klotho基因酵母双杂交诱饵载体的构建及其毒性与自激活效应检测 张疆 董素珍（脑功能基因组学教育部、上海市重点实验室，华东师范大学脑功能基因组研究所，上海 200062） 利用酵母双杂交系统，构建分泌型人Klotho基因(hsKL)酵母双杂交体系中的诱饵载体，对其进行鉴定，并检测其表达产物对酵母细胞有无毒性作用及对报告基因有无自激活作用。 运用PCR技术从pcDNA3.1-hsKL质粒中扩增出hsKL基因片段, 并克隆入pMD18-T载体，测序鉴定正确后利用EcoRI和BamHI双酶切，克隆入同样双酶切的酵母双杂交载体pGBKT7中，命名为pGBKT7-hsKL。经测序正确后，用醋酸锂法转化酵母菌AH109，在缺陷型培养基上观察pGBKT7-hsKL在AH109中的表达情况，检测诱饵蛋白对酵母细胞有无毒性作用及对报告基因有无自激活功能。克隆出的hsKL基因除了两个无义突变外,序列正确。构建的诱饵载体pGBKT7-hsKL测序验证序列完全正确，并且融合区域无移码突变。pGBKT7-hsKL转化酵母感受态在SD/-Trp平板上生长良好，而在SD∕-His∕-Trp∕-Leu平板上不能生长，表明诱饵质粒转化成功，且对报告基因无自激活作用。接种于液体培养基的表达pGBKT7-hsKL的AH109分裂旺盛，表明诱饵质粒对酵母细胞无毒性作用。成功构建出了能正确表达人分泌型KL、对酵母细胞无毒性和自激活作用的诱饵载体pGBKT7-hsKL，可以作为“诱饵” ，为下一步筛选hsKL相互作用的蛋白建立了基础。 :人分泌型Klotho；酵母双杂交；诱饵载体；自激活效应 ：Q782 ：A Construction and expression of “bait plasmid”containing human secreted form Klotho gene in yeast two-hybrid system ZHANG Jiang, DONG Su-zheng (Key Lab of Brain Functional Genomics，MOE＆STCSM, Shanghai Institute of Brain Functional Genomics, East China Normal University, Shanghai, China, 200062) Abstract: Objective To construct a bait vector containing human secreted form klotho gene (hsKL) in yeast two-hybrid system for downstream screening the proteins interacting with hsKL. Methods The DNA fragment coding hsKL was amplified by PCR with designed primers from the plasmid of pcDNA3. 1-hsKL and then cloned into pMD18-T vector. After verified with restriction endonuclease digestion of EcoRI and BamI, the right fragment of hsKL gene determined by sequencing was subcloned into the“bait plasmid”pGBKT7(named as pGBKT7-hsKL). After confirmation with restricted endonuclease digestion and sequence analysis, the plasmid was transformed into the yeast cell AH109, and its toxicity and transcriptional activation was tested by the phenotype assay. Results The amplified product of 1.6kb was inserted into pMD18-T Vector and proven correctly by double restriction enzyme digestion. Sequence analysis revealed that the fragment was correctly inserted into pGBKT7 with a right reading