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

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 poly-merase Ⅱ was amplified from the genomic DNA of Blepharisma japonicum. The PCR product digested by Bam H Ⅰ and Xho Ⅰ was cloned into expression vector pGEX-6pl digested by the same enzymes. The recombinant plasmid pGEX-6pl-ZFbl was transformed to E. coli strain BL21(DE3)and the GST-ZFbl fusion protein was expressed. The GST-ZFbl fusion protein was detected by Western blotting with anti-GST antibodies. New Zealand rabbit was injected with purified GST-ZFbl protein to induce immunoreactions. The polyclonal antibody was detected by ELISA and Western blotting, which indicated that highly reactive and specific antiserum was obtained and antiserum liter reached to 1:15000.