hsnha形成二聚体行使转盐功能.docVIP

破骨细胞钠氢转运蛋白关键位点研究 黄晓斌，仲蕾蕾 （400030重庆，重庆大学农学及生命科学研究院） [摘要]目的 在酿酒酵母中异源表达人破骨细胞分化成熟潜在因子钠氢转运蛋白2，对其关键氨基酸保守位点进行突变分析，鉴定其作为盐离子载体转运Na+的功能。 方法 采用突变试剂盒依次把此蛋白的D278和D279 2个位点的天冬氨酸中1个氨基酸突变成半胱氨酸，得到2个不同位点突变的定点突变体；构建酵母双基因表达载体，通过电穿孔的方式转入到酵母菌株中；Western印迹检测2个定点突变基因在酵母菌株的表达；通过NaCl选择压力显示2个突变体在高盐环境中的相互作用，观察菌株的抗盐生长表型。结果 成功获得适合转化酵母的多个载体；在固体和液体培养基中，经半乳糖诱导后，各个载体均可使酵母异源表达钠氢转运蛋白2；生长曲线显示2个突变体可相互作用，部分恢复酵母菌株的抗盐能力。 结论 钠氢转运蛋白2通过关键位点D278和D279 2，以形成二聚体的方式在细胞中发挥着钠离子转运功能。 [关键词] 破骨细胞；钠氢转运蛋白2；酿酒酵母 [中图法分类号] [文献标志码] A Key point of Na+-H+ antiporter to transport sodium in osteoclast Huang Xiaobin , Zhong Leilei (Institute of Agriculture and Life Science, Chongqing University, 400030,China) [Abstract] Objective Expressing heterologously Na+-H+ antiporter2 that potentially benefits the differentiation and maturation of the osteoclast in Saccharomyces cerevisiae, to directly mutate the conservative key amino acid and identify its function in transporting Na+ as a salt transporter. Methods In turn mutating one of the two Asp of the antiporter into Cys by mutation kit, to achieve two different mutants, to build two gene yeast vector that can expressed simultaneously in Saccharomyces cerevisiae and electroporate it into yeast. The expressing of the two antiporter mutants in the strain were detected by Western blot. Observe their interaction of the two mutants by the growth curve of BW31a in high salty concentration medium. Results Achieve several vectors to transform yeast successfully. Each vector can make the yeast express the Na+-H+ antiporter2 after inducing by galactose in the solid or liquid medium normally. The growth curve reveal that the two mutants can interacts to partly rescue the ability of yeast growing into high salt concentration medium. Conclusion The D278 and D279 sites is very important for Na+-H+ antiporter2, it transport sodium maybe through forming dimmer in cells to maintain their regular growth. [Key words] osteoclast; Na+-H+ antiporter2; sacc