大鼠ANT1蛋白的原核表达纯化及多克隆抗体的制备

Expression,purification of rat ANT1 protein and preparation of its polyclonal antibody

目的:表达和纯化大鼠腺苷酸转运体(ANT1)蛋白并制备其多克隆抗体.方法:通过PCR扩增大鼠脑组织中ANT1的编码区序列,构建ANT基因重组载pET28a-ANT1,转化至E. coli BL21(DE3),IPTG诱导表达,Ni2+-NTA离子交换树脂层析纯化;纯化的ANT1蛋白免疫新西兰大白兔制备多克隆抗体并亲和纯化,并通过Western Blot法鉴定其特异性.结果:成功构建重组质粒pET28a-ANT1,经IPTG诱导在大肠杆菌中表达了Mr约为32×103的目的蛋白;亲和层析纯化后免疫的新西兰大白兔获得其多克隆抗体;曝光后胶片上均可见Mr32×103的阳性条带,与纯化的大鼠ANT1重组蛋白处于同一水平,表明纯化的抗ANT多克隆抗体具有高度的特异性.结论:利用基因重组技术,在大肠杆菌中成功表达了大鼠ANT1融合蛋白并制备了其多克隆抗体,为研究腺苷酸转运体蛋白在癫痫发病过程中的作用机制奠定了基础.

AIM：To express and purify the adenine nucleotide transporte protein （ANT1）and to prepare its polyclonal antibody. METHODS：The CDS of ANT1 was amplified by PCR from rat,and then subcloned into prokaryotic expression vector pET28a ,after transformed with the vector,the BL21（DE3）stains were induced by IPTG. The expression product was purified by Ni^2＋-NTA ion exchange resin. The purified protein was analyzed by SDS-PAGE,and then immuned the New Zealand white rabbits to prepare antibody. The polyclonal antibody was received by Affinity purification from rabbit serum. The prepare antibody was used in sample assay by Western Blot. RESULTS： The expression plasmid pET28a-ANT1 was constructed successfully, and rat ANT1 with 32 000 molecular weight was expressed in E. coli. The purity of the ANT1 protein was purification with Ni^2＋ -NTA ion exchange resin and then immuned the New Zealand white rabbit to prepare antibody. It was comfirmed by Western Blot technique that antibody specifically recognized the 32 000 ANT1 protein. CONCLUSION： We had successfully obtained prokaryotic fusion protein of ANT1, got high sensitivity and specification anti-ANTI polyclonal antibody. That lays a solid foundation for the studies of ANT1 in pathogenesis of epilepsy.