凋亡素原核表达载体的构建、表达与抗体制备
onstruction of prokaryotic expression vector for Apoptin and Preparation of Antibody of Apoptin
目的：构建凋亡素基因的原核表达载体，进行表达并制备表达蛋白的多克隆抗体。方法：构建凋亡素pGEM-T/Apoptin载体，并将凋亡素基因亚克隆至原核表达载体pET-28a(+)中，将该质粒转化至大肠杆菌E.coli BL21(DE3)受体菌中，以IPTG对其进行诱导表达，聚丙烯酰胺凝胶电泳分析目的蛋白。用表达蛋白常规免疫SD大鼠，间接ELISA检测大鼠血清中抗体的滴度。结果：凋亡素基因被成功的克隆至pET-28a(+)，表达产物经SDS-PAGE分析，在相对分子量约17 000的位置出现目的蛋白条带，大小与预期结果一致，SD大鼠经五次免疫后，得到了滴度为1：5×105血清中和抗体。结论：凋亡素原核表达载体的成功构建和多克隆抗体的制备为进一步研究凋亡素的功能和凋亡素的应用研究奠定了基础。
Objective To construct the prokaryotic expression vector for Apoptin and do the preparation of antibody of Apoptin. Methods The Apoptin gene from was amplified from pGEM-T/Apoptin plasmid by PCR and cloned into pET-28a (+). E.coli BL21 (DE3) were transformed by the recombinant plasmid pET-28a (+) and induced by IPTG, then the protein was analysised by SDS-PAGE. SD rats were immunized with the protein and the titre of antibody was determined by indirect ELISA. Results The Apoptin gene was successful cloned to pET-28a (+), the specific protein had an apparent related molecular weight of about 17 000 as indicated by SDA-PAGE analysis. The antibody titre in blood stream reached to 1：5×105 after fifth immunizations. Conclusions The prokaryotic expression vector for Apoptin was successful constructed and the antibody was prepared, which settled a foundation for further study of the function and application of Apoptin.
杨广笑、王健生、王全颖
分子生物学生物工程学生物化学
凋亡素蛋白原核表达抗体制备
poptinprokaryotic expression vectorantibody preparation
杨广笑,王健生,王全颖.凋亡素原核表达载体的构建、表达与抗体制备[EB/OL].(2008-10-27)[2025-08-02].http://www.paper.edu.cn/releasepaper/content/200810-671.点此复制
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