摘要
目的克隆zeta链蛋白基因并在大肠杆菌中表达,经纯化与鉴定,为建立α-SEA型地中海贫血的免疫学筛查方法提供特异性抗原。方法从经处理的K562细胞中提取总RNA,采用RT-PCR技术反转录并将其克隆到pET-21a(+)载体中,测序验证。融合蛋白在大肠杆菌中表达,采用Probond树脂柱纯化融合蛋白,用WesternBlotting和ELISA鉴定。结果成功构建了融合表达载体pET-zeta,融合蛋白得到可溶性表达及纯化,经Westernblotting和商品ELISA试剂盒鉴定显示重组蛋白为zeta链蛋白。结论克隆表达并获得了纯化zeta链蛋白,为地中海贫血的免疫筛选方法的建立提供了抗原。
Objective To obtain lmman zeta chain protein by gene cloning and expression in E. Coli, which would be used as specific antigen in preparation of ELISA kit for detection of zeta chain. Methods The gene of zeta chain was amplified by RT-PCR, then cloned into pET-21a（ ＋ ） vector, which was confirmed by DNA sequencing. The recombinant zeta chain as form of fusion protein was expressed in E. coli, purified by Probond resin column, and identified by Western blotting and ELISA. Results Fusion expression vector of pET-zeta was constructed; the fusion protein was effectively expressed in soluble form; and purified protein was shown a correct molecular weight and antigenicity of zeta hemoglobin confirmed by Western Blotting, Irranunol Dot and conanercial ELISA kit. Condusion Recombinant zeta chain was prepared successfully, which would be used in estahlishment of immunologic method to screen the α^-SEA thalasseimia.
出处
《中国实验诊断学》
2005年第4期491-494,共4页
Chinese Journal of Laboratory Diagnosis
基金
广东省重大课题资助项目:2004-A30801003
关键词
ZETA
克隆
表达
recombinant zeta hemoglobin
gene cloning
expression
