ΦC31整合酶的表达、纯化及多克隆抗体的制备

Expression,purification and polyclonal antibody preparation of ΦC31 integrase

目的:提供ΦC31整合酶研究所需的大量纯化蛋白质及特异性抗体。方法:将编码ΦC31整合酶的ORF插入到pET22b+载体构建表达质粒并转化E.coliBL21(DE3),用IPTG诱导表达His-C31整合酶融合蛋白,His-Bind Quick Car-tridge纯化表达产物。重组蛋白作为抗原常规免疫家兔制备相应的多克隆抗体,Western blotting确定抗体的特异性,体外线性底物分子内重组检测系统检测抗体的活性。最后用该抗体检测了ΦC31整合酶在真核细胞HEK293中的定位和分布。结果:16℃条件下0.2 mmol/LIPTG诱导培养30 h,携带pET22b-ΦC31质粒的E.coliBL21(DE3)在YTG培养基中,可表达出大量的融合蛋白,该蛋白经体外线性底物分子内重组检测系统检验具有较高的活性。使用该融合蛋白免疫新西兰大白兔后,分离兔血清可得到ΦC31整合酶多克隆抗体,Western blotting和荧光免疫细胞组织化学方法证实,该抗体可特异性地识别细菌及细胞表达的ΦC31整合酶蛋白抗原。结论:本实验建立了表达ΦC31整合酶的方法,并成功制备了ΦC31整合酶特异性抗体。

Aim： To prepare pure ΦC31 integrase and its specific polyclonal antibody. Methods： The ORF of ΦC31 integrase was inserted into pET22b＋ to construct ΦC31 integrase expression vectors （pET22b-ΦC31）. The His-tag fusion protein was expressed through IPTG inducement in E. coli BL21 （DE3） and purified with His-Bind Quick Cartridge. The purified fusion protein was used as an antigen to immune rabbits to produce polyclonal antibody in routine. The specificity of the antibody was verified by Western blotting, and in situ immune was anaylzed by in vitro linear intramolecular assay system. The location and distribution of ΦC31 integrase in C31 integrase expressed HEK293 cells were further determined by fluorescence assay. Results： The BL21 （DE3） pET22b-ΦC31 expressed abundant fusion protein in YTG culture medium after being induced by IPTG 0. 2 mmol/L for 30 hours. The specific polyclonal antibody against ΦC31 integrase was obtained from the sera of the His-ΦC31 integrase fusion protein immunized rabbits. Furthermore, this kind of protein was proved to have higher recombination activity based on the in vitro approach, Conclusion： The ΦC31 integrase recombination protein could be obtained through the structed ΦC31 integrase express system, and ΦC31 integrase specific antibody could be produced by immunized rabbits with the fusion protein.