Cloning And Studying Of A Novel Zinc Finger Protein Gene ZNFD

Zinc finger proteins are among the most abundant proteins in mammals, which contained the zinc finger motifs. A novel human gene ZNFD, gene ID NM₁82761, which was not been investigated. The bioinformatics character suggested that ZNFD is a member of zinc finger protein. The main complishments are as follows.In this work, we cloned the ZNFD gene from testis, then identified by sequence. Bioinformatics databases were used to analyze and predict its function. We confirmed that ZNFD gene included a 1432bp nucleotide sequence, has a 990bp full open reading frames (ORFs), which was localized on chromosome 5q23.1. It was composed of five exons and four introns. The ZNFD gene encoded a protein with 329 amino acids. The molecular weight and isoelectric point were predicted to be 37KD and 5.0, respectively, using software. In order to investigate the tissue distribution of ZNFD, the expression pattern of ZNFD was determined by PCR amplification use a cDNA panel of eighteen human tissues, purchased from Clontech Instituts. The experiment suggested that ZNFD was expressed in prostate gland, brain, pancreas, uterus, specifically has a high expression in testis. To construct the recombinant green fluorescence expression vector pEGFP-ZNFD, then transiently transfected into COS-7 cell. We found that the strongest green fluorescence concentrated in nucleus. In our experiment, we detect the subcellular location of ZNFD protein is in nucleus.Secondly, prepare and obtain ZNFD polyclonal antibody. Pattern of ZNFD ORFs sequence was choosen by DNAstar software, was cloned into pET-32a prokaryotic expression vector to form pET32a-ZNFD plasmid. His-ZNFD protein was expressed in E.coli Rosetta-gami(DE3) inducing by IPTG, 45kDa molecular weight protein can be expressed with a high efficiency, then purified by using affinity chromatography. Purified His-ZNFD protein was used to immunize rabbits to prepare polyclonal antibody. Rabbit serum specificity and its titer were detected by double diffusion and western blot, respectively. The data indicated titer already 1:32 and the polyclonal antibody had high antigenicity.Eventually, has constructed the recombinant eukaryotic expression pcDNA-TAP-ZNFD to transfect into 293T cell. Cell lysate were analyzed by Western blot to determine the expression of ZNFD protein.In this work, we cloned the ZNFD gene. Bioinformatics database and software were used to analyze and predict its function. Then RT-PCR analysis showed that ZNFD was expressed in several tissues. Green fluorescence protein localization showed that ZNFD located nucleus. Obtained the purified fusion protein ZNFD, it has high antigenicity. Prepared polyclonal antibody that provided a reliable tool for the future research.The pcDNA3-TAP vector is used for tandem affinity purification(TAP), which is a rapid and reliable technique that can be successfully applied in the analysis of protein-protein interaction networks in prokaryotic and eukaryotic cells under close-to-physiological conditions.