花背蟾蜍转录因子Pax6 variant的原核表达和多克隆抗体制备
Prokaryotic expression and polyclonal antibody preparation of the transcription factor Pax6 variant of Bufo raddei Strauch
目的：构建花背蟾蜍（Bufo raddei Strauch）转录因子Pax6 variant的原核表达载体，纯化融合蛋白His-Pax6 variant并以其为抗原免疫家兔, 制备花背蟾蜍转录因子Pax6 variant多克隆抗体。 方法：用RT-PCR扩增出花背蟾蜍胚胎组织中的Pax6 variant 基因，并将片段与原核表达载体pET-28a-c 连接, 构建重组表达质粒pET-Pax6 variant并转化大肠杆菌Rosetta(DE3)菌株,诱导融合蛋白His-Pax6 variant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Pax6 variant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43 300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Western blot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Pax6 variant 蛋白及特异性多克隆抗体，对进一步研究花背蟾蜍Pax6 variant在发育过程中功能奠定基础。
m: 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, 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.
张蕊、宋德潇、高岚、李杰
分子生物学生物工程学遗传学
花背蟾蜍Pax6 variant原核表达多克隆抗体
Bufo raddei StrauchPax6 variantProkaryotic expressionpolyclonal antibody
张蕊,宋德潇,高岚,李杰.花背蟾蜍转录因子Pax6 variant的原核表达和多克隆抗体制备[EB/OL].(2008-09-26)[2025-08-16].http://www.paper.edu.cn/releasepaper/content/200809-774.点此复制
评论