The Gene Cloning And Expression Of Human E2F3

Objective: E2F family of transcription factors are key regulators from stage of G1 to S in the cell cycle. Meanwhile, E2F factors have tightly relationship with the occurrence of tumor and apoptosis. In order to study their structures, functions and their relationship with the occurrence of tumor, we has cloned E2F3 gene from human tissue, constructing prokaryotic expression vector and eukaryotic expression vector and realized its prokaryotic expression and eukaryotic expression, so we have made firm basis for further researchMethods: We extracted total RNA of tumor cell from human liver by Trizol method, then reverse transcribed RNA to cDNA. E2F3 gene was cloned by nested-PCR and overlap-PCR using cDNA as template. Then we constructed 4 prokaryotic expression vectors of E2F3 gene, which are pET28a-E2F3, pET32a-E2F3, pQE30-E2F3, and pGEX-4T-E2F3 and a eukaryotic expression vector, pEGFP-Nl-E2F3. BL21 engineering bacteria is transformed by prokaryotic expression vector and induced to express the recombinant E2F3 protein Moreover, we detected the recombinant protein by Western blotting. We transfected T24 cell by eukaryotic expression vector, observing whether or not E2F3 gene has been expressed in eukaryotic cell.Results: the full-length of E2F3 gene has been successfully cloned. We have realized prokaryotic expression of E2F3 gene by changing expression vectors and tested recombinant protein expressed by Western blotting. In addition, we constructed eukaryotic expression vector of E2F3 gene. Transfection of T24 cell shows that E2F3 gene has expressed in eukaryotic cell. This result sets up a foundation for further experiments of cytologyConclusion: The part of E2F3 gene, which is near 5' end, is GC-rich. This part greatly affects PCR amplification of E2F3 gene. It is supposed that this sequence is an internally adjustable area of E2F3 gene. It maybe forms hairpin structures that regulate expression of E2F3 gene. In addition ,We have discovered that the expression of E2F3 gene is different in different prokaryotic expression vector and the fusion of GST and E2F3 gene is much easier to realize prokaryotic expression. This may becaused by difference in functional element of expression vector and features of E2F3 gene itself...