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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。
OBJECTIVE: To heterologously express human osteoclast differentiation and maturation factor sodium hydrogen transport protein 2 in Saccharomyces cerevisiae, and to analyze the mutation of its conserved key amino acid sites and to identify its function as a salt ion transporter for Na + transport. Methods One amino acid in aspartic acid at D278 and D279 of the protein was mutated to cysteine by mutagenesis kit, and two site-directed mutants with different site mutations were obtained. The expression vectors were transformed into yeast strains by electroporation. Western blotting was used to detect the expression of two site-directed mutants in yeast strains. The pressure-selective NaCl stress showed the interaction of two mutants in high salt environment. Salt-resistant growth phenotype. The results showed that several vectors were successfully obtained, which could be transformed into yeast. In both solid and liquid media, each vector could induce yeast heterologous expression of Na + 2 after induced by galactose. The growth curve showed that the two mutants could interact , Partially restore the salt resistance of yeast strains. Conclusion Sodium transporter 2 plays a role of sodium ion transport in cells through the key sites D278 and D279 2, forming dimers.