小鼠RMND5A的原核表达、纯化及多克隆抗体制备

RMNDSA通过MAPK信号途径参与了对细胞生命活动的调控过程.通过PCR扩增RMND5A基因,并将其克隆至表达载体pGEX-4T-1上,转化至大肠杆菌BL21（DE3）中,再通过IPTG进行诱导表达GST-RMND5A融合蛋白.通过尿素洗涤包涵体并切胶回收纯化融合蛋白,免疫新西兰大白兔制备多克隆抗体,Western blot检测抗体活性.结果说明,获得了RMND5A原核表达重组融合蛋白及高效价的特异性兔抗RMND5A多克隆抗体,为RMND5A进一步的功能研究奠定了基础.

Prokaryotic Expression,Purification of Mouse RMND5 A Protein and Preparation of its Polyclonal Antibody

RMND5 A is involved in regulation of cell life progress through acting a role in MAPK pathway. A fragment of RMND5A gene encoding region was amplified by PCR. The PCR product was cloned into the expression vector pGEX- 4T-1 and transformed into Escherichia coli BL21 （DE3）. The BL21 （DE3） strain, containing RMND5A recombinant plasmid, was induced with IPTG （isopropylthio-β-D -galactoside）. The fusion protein was purified by UREA and separated by SDS-PAGE and recovered by gel extraction. White rabbits originated in New Zealand were immunized with the separated protein. The antibody was detected by Western blot. The result was showed that the GST fusion protein of RMND5A and specificity anti-RMND5A polyclonal antibody had been successfully obtained. That lays a solid foundation for the further research of RMND5A function.