摘要
(1)目的构建α-4贾第素诱饵蛋白质粒,为贾第素功能的研究奠定基础。(2)方法从α-4贾第素克隆载体pGM-T-α-4中双酶切获得α-4贾第素编码序列,与酵母表达质粒pGBKT7在DNA连接酶的作用下进行重组,重组载体通过热休克法转化大肠杆菌感受态细胞,大肠杆菌经过培养、筛选,碱裂解法提取质粒酶切鉴定获得pGBKT7-α-4诱饵载体。并对诱饵载体进行自激活验证,利用Western blot鉴定诱饵蛋白的表达情况。(3)结果经酶切和测序鉴定,获得了pGBKT7-α-4诱饵载体。转化子Y187酵母细胞在SD/-Trp平板上能够生长;而在SD/-Trp/-His和SD/-Trp/-Ade平板上不能生长。说明pGBKT7-α-4质粒无自我激活作用。Western blot鉴定结果显示,pGBKT7-α-4转化酵母菌在52KD水平位置可检测到α-4融合C-Myc蛋白的表达。(4)结论构建α-4贾第素诱饵蛋白质粒。诱饵蛋白质粒无自我激活作用,在酵母菌株中表达良好,为贾第素功能的研究奠定了基础。
Objective Construction ofα-4 giardin decoy protein particles.In order to study the function of giardin.Methods Theα-4 giardin encoding sequence was obtained by double enzyme digestion from theα-4 giardin cloning vector pGM-T-α-4.Under the action of DNA ligase,theα-4 giardin coding sequence was combined with the yeast expression plasmid pGBKT7.The recombinant plasmid transformed the competent cells of escherichia coli by heat shock.Coli was cultured and screened.Finally,the plasmid was extracted by alkaline lysis and identified by enzyme digestion.The pGBKT7-α-4 decoy vector was obtained.In addition,the self activation capability of bait vector was verified,and the expression of bait protein was identified by wstern blot.Results The pGBKT7-alpha-4 decoy vector was obtained by restriction enzyme digestion and sequencing.Transformants Y187 yeast cells could grow on SD/-Trp plates,but not on SD/-Trp/-His and SD/-Trp/-Ade plates.Therefore,there was no self activation of pGBKT7-α-4 plasmid.Western blot identification showed that the expression ofα-4 fusion C-Myc protein could be detected at the level of 52 KD in pGBKT7-α-4 transformed yeast.Conclusion Successful construction ofα-4 giardin decoy protein particles.The bait protein granules show no self activation and are well expressed in yeast strains.It lays a foundation for the study of giardin function.
出处
《华北理工大学学报（医学版）》
2017年第6期421-425,439,共6页
Journal of North China University of Science and Technology：Health Sciences Edition
基金
国家自然科学基金(编号:31471954)
关键词
α-4贾第素
构建诱饵蛋白质粒
酵母双杂交
α - 4g i a r d i n. C o n s t r u c t i o no fb a i tp r o t e i np a r t i c l e s . Y e a s t t w oh y b r i d
