跨膜逆向转运蛋白NHXFS1多克隆抗体的制备及应用

NHXFS1基因是通过DNA家族改组（DNA family shuffling）技术,以拟南芥、水稻和菊花的液泡膜Na＋/H＋逆向转运蛋白基因（NHX1）为亲本获得的活性显著增强的新基因。为制备该蛋白的多克隆抗体,对该蛋白进行跨膜结构分析,选取跨膜蛋白的C末端为靶标,并将其克隆到原核表达载体pET32a中,成功构建了原核融合蛋白pET32a-NHXFS1-抗原表达载体,转化大肠杆菌BL21（DE3）并诱导表达。通过镍柱亲和层析纯化该融合表达蛋白,获得了纯度约为80%的纯化蛋白,用于免疫新西兰大白兔制备多克隆抗体。ELISA实验表明,该抗体的效价达到1：128 000,提取表达NHXFS1蛋白的酵母液泡经该多克隆抗体Western blot检测,证明该抗体具有较好的NHXFS1蛋白特异性。NHXFS1多克隆抗体的制备为进一步认识NHXFS1新蛋白结构与功能以及植物耐盐分子生物学的研究奠定了基础。

The Preparation and Application of NHXFS1 Polyclonal Antibody

NHXFS1 was a novel powerful vacuolar Na^＋/H^＋ antiporter gene which was obtained from Arabidopsis thaliana antiporter gene AtNHX1, Oryza sativa antiporter gene OsNHX1 and Dendranthema morifolium antiporter gene DmNHX1 by DNA family shuffling technology. In this study, we prepared and identified the polyclonal antibody against the C-terminal fragment of NHXFS1. For expression and purification of NHXFS1-C terminal protein, the expression vector pET32a-NHXFS 1 antigen containing 6xHis tag was constructed and trans- ferred into the E.coli strain BL21 （DE3）. After IPTG induced, the fusion protein was purified by Ni-NTA superflow cartridges and was immunized with rabbits for polyclonal antibody preparation. The ELISA results showed that the potency of polyclonal antibody reached 1：128 000. To identify the antibody specificity, the vacuolar was extracted from the yeast stain expressing NHXFS1 and OsNHX1 protein and analyzed by Western blot. The result indicated that we have obtained high specificity polyclonal antibody that could be used for further study of Na^＋/H^＋ antiporter and the molecular mechanism of plant salinity.