赭纤虫RNA聚合酶Ⅱ锌指基因片段的表达、纯化及多克隆抗体的制备

THE EXPRESSION AND PURIFICATION OF RNA POLYMERASE Ⅱ ZINC FINGER GENE FRAGMENT FROM BLEPHARISMA JAPONICUM AND PREPARATION OF POLYCLONAL ANTIBODY

以一种进化较为原始的单细胞真核生物日本赭纤虫(Blepharisma japonicum)大核基因组DNA为模板,PCR扩增 得到了RNA聚合酶锌指基因片段,并构建重组表达质粒pGEX-6p1-ZFbl,在大肠杆菌BL21(DE3)中进行表达,SDS- PAGE和Western blot分析证明目的蛋白得到了可溶性融合表达。用纯化的蛋白免疫新西兰兔,制备多克隆抗体。 Western印迹和ELISA鉴定结果表明抗体特异性较高,效价高达1:15000。

Zinc finger protein is the largest and most diverse superfamily of nucleic acid binding proteins in eukaryotes. Zinc finger has a unique structure requiring a zinc ion in the core with several amino acid residues, which are cysteins or histidines in most cases. Zinc fingers are kinds of transcription factors. The role of zinc finger of RNA Polymerase Ⅱ is binding special DNA sequence and activates transcription. Blepharisma japonicum is a lower unicellular eukaryote. The cell contains two nuclei, the transcriptionally quiescent micronucleus and the transcriptionally active macronucleus. In order to study the mechanism of the transcription regulation in lower eukaryotes, zinc finger gene fragment of RNA polymerase Ⅱ was amplified from the genomic DNA of Blepharismajaponicum. The PCR product digested by BamH Ⅰ and XhoⅠ was cloned into expression vector pGEX-6p1 digested by the same enzymes. The recombinant plasmid pGEX-6p1-ZFb1 was transformed to E. coli strain BL21（DE3） and the GST-ZFb1 fusion protein was expressed. The GST-ZFb1 fusion protein was detected by Western blotting with anti-GST antibodies. New Zealand rabbit was injected with purified GST-ZFb1 protein to induce immunoreaetions. The polyelonal antibody was detected by ELISA and Western blotting, which indicated that highly reactive and specific antiserum was obtained and antiserum titer reached to 1 ： 15000.