| 李杰,高岚,宋德潇.Pax6 variant新基因的原核表达蛋白纯化和多克隆抗体制备[J].济宁医学院学报,2011,(6):381-384,402 |
| Pax6 variant新基因的原核表达蛋白纯化和多克隆抗体制备 |
| Prokaryotic expression,affinity protein purification and polyclonal antibody preparation of a Novel Gene Pax6 variant |
| 投稿时间：2011-11-01 |
| DOI：10.3969/j.issn.1000-9760.2011.06.001 |
| 中文关键词: 花背蟾蜍|Pax6 variant|原核表达|多克隆抗体 |
| 英文关键词: Bufo raddei Strauch|Pax6 variant|Prokaryotic expression|polyclonal antibody |
| 基金项目: |
| 作者 | 单位 | | 李杰 | 济宁医学院精神卫生学院, 山东济宁 272067 | | 高岚 | 兰州大学生命科学学院, 甘肃兰州 730000 | | 宋德潇 | 兰州大学第一人民医院, 甘肃兰州 730000 |
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| 中文摘要: |
| 目的 构建花背蟾蜍(Bufo raddei Strauch)转录因子Pax6variant的原核表达载体,纯化融合蛋白His-Pax6variant并以其为抗原免疫家兔,制备花背蟾蜍转录因子Pax6variant多克隆抗体。方法 RT-PCR扩增得到花背蟾蜍胚胎组织的Pax6variant基因,并将此片段与原核表达载体pET-28a-c连接,构建重组表达质粒pET-Pax6variant并转化大肠杆菌Rosetta(DE3)菌株,诱导融合蛋白His-Pax6variant表达;所获得的可溶性蛋白经亲和层析纯化、SDS-PAGE电泳鉴定后,免疫家兔制备抗血清,分别采用ELISA、Western blot检测抗体效价和特异性。结果 从花背蟾蜍胚胎组织中克隆了Pax6variant基因,并成功构建了其原核表达质粒;SDS-PAGE结果证实获得Mr为43300的His-Pax6融合蛋白且为可溶性蛋白;经过His亲和层析有效纯化;以该融合蛋白免疫家兔制备得到的抗血清经Westernblot检测证实能与目的蛋白发生特异性结合,ELISA检测为阳性。结论 获得了花背蟾蜍转录因子Pax6variant蛋白及特异性多克隆抗体,对进一步研究花背蟾蜍Pax6variant在发育过程中功能奠定基础。 |
| 英文摘要: |
| Objective To construct the prokaryotic plasmid expressing the transcription factor Pax6 variant of Bufo raddei Strauch, purify His-Pax6 variant fusion protein produced by the expression system, and prepare its antiserum. Methods The transcription factor Pax6 variant was amplified by RT-PCR from the embryo of Bufo raddei Strauch and cloned into prokaryotic expression vector pET-28a-c, and then expressed in E.coli Rosetta(DE3) in which His-Pax6 fusion protein was induced by IPTG.After the soluble protein was purified by His affinity chromatography and identified by SDS-PAGE, the rabbits were immunized with the fusion protein and the antiserum was obtained.And the sensitivity and specificity of the antibodies were evaluated by enzymelinked immunosorbent assay(ELISA) and Western blot analysis. Results The transcription factor Pax6 of Bufo raddei Strauch was cloned and sequenced.Its recombinant protein was obtained and polyclonal antibody was prepared. Conclusion Pax6 protein and its specific polyclonal antibody have been prepared, which provides a basis for the establishment of immunoassays of Bufo raddei Strauch Pax6. |
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