人类心脏和肌肉组织特异表达基因Smyd1的表达与抗体制备

Expression of a Heart and Muscle-specific Expressed Smyd1 Gene in E. coli and Preparation of Its Antibody

Smyd1基因是人类心脏和肌肉特异表达基因,制备该基因的多克隆抗体可以为进一步深入研究Smyd1在心脏发育过程中与其他因子相互作用提供检测工具.通过PCR方法扩增smyd1基因的编码区片段,并将其克隆至PGEX-4T-1上,转化到大肠杆菌BL21(DE3)中,再通过5052法和IPTG法分别诱导表达GST-Smyd1融合蛋白.经比较,5052法诱导的蛋白表达量明显高于IPTG法.采用5052法大量诱导表达,通过割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.结果表明,实验获得了高质量的多克隆抗体.

The Smydl gene is specially expressed in human heart and muscle tissues. The muhi-clonal antibody of Smydl protein is essential in further studies on the interaction of Smydl with other factors during the development of heart. A fragment of Smydl 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 Smydl recombinant plasmid, was induced with 5052 and IPTG （isopropyhhio-/3-D-galactoside）. After comparison with the two methods, 5052 was selected as the one to induce massively because of its higher efficiency expressed. The fusion protein was separated on SDS- PAGE and recovered by gel extraction. New Zealand big white rabbits were immunized with the separated protein. The antibody was detected by Western blot. The result showed that the high quality muhi-clonal antibody was obtained.