| 果蝇中Ecp蛋白的表达、纯化与抗体制备 |
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| 引用本文： | 汪道涌,刘莉,谢维.果蝇中Ecp蛋白的表达、纯化与抗体制备[J].生物工程学报,2003,19(3):291-293. |
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| 作者姓名： | 汪道涌 刘莉 谢维 |
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| 作者单位： | 东南大学遗传与发育生物学系,遗传学研究中心,南京,210009 |
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| 基金项目： | 教育部高等学校优秀青年教师教学科研奖励计划基金资助 (No .2 0 0 1 182)~~ |
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| 摘 要： | 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DH 5α，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Western Blot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，提示ecp可能是一个重要的管家基因。
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| 关 键 词： | ecp， 克隆， 表达， 抗体制备， 黑腹果蝇 |
| 文章编号： | 1000-3061(2003)03-0291-03 |
| 修稿时间： | 2002年11月18 |
Preparation of Antibodies Against a Novel Leucine-zipper Protein, Ecp |
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| WANG Dao,Yong,LIU Li,XIE Wei.Preparation of Antibodies Against a Novel Leucine-zipper Protein, Ecp[J].Chinese Journal of Biotechnology,2003,19(3):291-293. |
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| Authors: | WANG Dao Yong LIU Li XIE Wei |
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| Institution: | Genetics Research Center, Department of Genetics and Developmental Biology, Southeast University, Nanjing 210009, China. |
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| Abstract: | 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 5alpha. 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. |
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| Keywords: | Ecp cloning expression antibody preparation Drosophila melanogaster |
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