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Initial Exploration of ZNFD Biological Functions and ZBTB9 Cloning and Expression of Mouse Zinc Finger Protein

Author: XuFengQin
Tutor: XuWeiAn
School: Suzhou University
Course: Zoology
Keywords: Zinc finger protein ZNFD HSE Transcriptional activation ZBTB9
CLC: Q78
Type: Master's thesis
Year: 2011
Downloads: 7
Quote: 0
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Abstract


C 2 H 2 -type zinc finger protein is important zinc finger protein family members, as a transcription factor, broad participation in transcriptional regulation, cell proliferation, differentiation and apoptosis other life activities. Our laboratory by RT-PCR from mouse tissues obtained two mouse genes ZNFD (NM 0 01004061) and ZBTB9 (NM 0 01005916), SMART analysis showed that the structure of domain , ZNFD protein containing a C-terminal C2H2 zinc finger motif, ZBTB9 protein N-terminal BTB domain containing, C terminal containing two C2H2 zinc finger motif, which shows both C2H2 zinc finger protein family . About ZNFD and ZBTB9 functional studies in mice have been reported yet, so these two genes in our laboratory started initial research. (1) First, by RT-PCR method from mouse testis cDNA obtained ZNFD gene sequencing results verify ZNFD sequence accuracy. Using bioinformatics approach to ZNFD genes and proteins were analyzed, the results showed that, ZNFD located on chromosome 18, located in the 18qD1. The gene contains a 1002bp open reading frame encoding a 333 amino acid composition, molecular weight of 37.4kDa protein. Organized on ZNFD expression analysis confirmed ZNFD in mouse testis-specific expression, and the expression level is higher. Expression analysis shows ZNFD time about 13 days in the mouse was born around expression. Right ZNFD in seven species homology analysis showed that, ZNFD different high homology between species. By building green fluorescent protein fusion expression vector pEGFP-ZNFD its subcellular localization analysis showed ZNFD localized in the nucleus. To identify ZNFD protein N-terminal domain and the zinc finger domain of its nuclear localization effects, build a series of truncated ZNFD with green fluorescent protein fusion recombinant plasmid Asian localization analysis showed ZNFD protein N-terminal amino acids ( 1-214) is required for its nuclear localization, and the zinc finger domain of ZNFD afford a key role in nuclear localization. Application of Dual-Luciferase reporter assay system ZNFD in AP1 (PMA), HSE, GRE, NF-κB, AP1, CRE, SRE, p53 signaling pathway analysis of transcriptional regulation showed that ZNFD mediated by the HSE activation of signaling pathways has obvious effect, and this activation in a dose-dependent manner. Further ZNFD different functional sections showed transcriptional regulation of the HSE ZNFD proteins play its role in transcriptional activation element needs to maintain its structural integrity. pHSE-Luc plasmid containing the HSE elements, the plasmid was designed to detect heat shock factor (HSF) and activation of heat shock protein-mediated transcriptional regulation of signaling pathways. HSE elements are typically present in the heat shock protein gene promoter region, transcription factors binding to the HSE element then the start of the downstream gene expression of heat shock protein. This study shows ZNFD may be used as heat shock protein-mediated signaling pathway regulates cell transcriptional activator to a series of biological functions. (2) bioinformatics analysis showed that mice ZBTB9 gene contains an open reading frame of 1380bp, encoding 459 amino acids. Start codon ATG, termination codon TGA. The gene encoded protein molecular weight of 49.5kDa. Chromosomal location for ZBTB9 analysis shows, ZBTB9 located on mouse chromosome 17, located in 17qA3.3. Subcellular localization analysis found ZBTB9 positioned in the nucleus and patchy distribution. By building ZBTB9 prokaryotic expression vector, in a prokaryotic expression system induces the expression of the fusion protein, and protein was purified to give the purified fusion protein further provides a polyclonal antibody preparation materials.

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CLC: > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)
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