果蝇中Ecp蛋白的表达、纯化与抗体制备

Preparation of Antibodies Against a Novel Leucine-zipper Protein, Ecp

获得特异性抗Ecp多克隆抗体 ,为进一步研究ecp的功能奠定基础。利用特异引物 ,通过RT PCR扩增出编码Ecp蛋白的全长cDNA ,克隆至谷胱甘肽S转移酶融合蛋白表达载体pGEX 4T 1(His) 6C中 ,转染大肠杆菌DH5α,经诱导表达后 ,利用谷胱甘肽琼脂糖珠从细胞裂解物中特异吸附融合蛋白 ,经凝血酶裂解 ,释放出Ecp蛋白 ,再经Ni NTA亲和层析 ,最终获得高纯度的Ecp蛋白。用纯化的Ecp蛋白免疫新西兰家兔 ,亲和层析纯化抗Ecp抗体。利用该抗体进行的WesternBlot结果表明 :Ecp蛋白在野生型黑腹果蝇胚胎、三龄幼虫神经系统、成虫、成虫头部组织中均有明显表达 。

The aim of this research is to prepare high quality polyclonal antibodies against Ecp, a recently identified leucine zipper protein. The full length cDNA of ecp was amplified by PCR, cloned into pGEX 4T 1(His) 6 and transformed into E.coli DH 5α. After induction with IPTG, the GST Ecp fusion protein from the lysate was bound to glutathione Sepharose 4B and digested with thrombin. The released Ecp protein was further purified through Ni NTA affinity chromatography to homogeneity. A rabbit was immunized with the purified Ecp, and the antibody generated against Ecp was purified by affinity chromatography. The results of the Western blot showed that Ecp is present in various development stages of Drosophila melanogaster, from larvae to adult.