ZBTB8A Polyclonal Antibody Preparation And Its Related Information Preliminary Study

Because the great number of members of ZBTB family mainly displaystranscription factors, mediated interaction between protein and protein or the specificbase sequences by its BTB domain and ZNF domain is to regulate gene transcriptionactivity, thus affecting important physiological functions on cell proliferation,differentiation, migration, apoptosis and cell cycle. Currently, there are seldom reportson ZBTB8A study. According to ZBTB8A information in the NCBI database, design aseries of primers. The ZBTB8A gene was cloned from AGS cells cDNA by RT-PCR andidentified by sequencing. Using the template of the identified sequences, a series offull-length and truncated ZBTB8A recombinant plasmids of various vectors wereconstructed in order to prepare for later experiments.To explore the subcelluar localization of ZBTB8A protein, GFP fusion expressionvectors of full-length and truncated ZBTB8A were constructed, which express the fusedprotein EGFP-ZBTB8A. The localization of ZBTB8A will be known by knowing thelocalization of EGFP. The results indicate that ZBTB8A was localized to the nucleus,forming dot-like structures. Further study shows that the BTB domain was required forspots like localization, and the ZNF domain was required for nuclear localization of theZBTB8A protein, thus implying that ZBTB8A protein exists in the nucleus as atranscription factor, of which both BTB domain and ZNF domain may play a key role innuclear localization of the ZBTB8A protein, which may be related to the functions oftheir own domains.To explore that ZBTB8A protein may be involved in the regulation of cellsignaling pathways because most proteins of ZBTB family are transcription factors,using some luciferase reporter vectors associated with cell signaling pathways is todetect the potential transcriptional regulation function of ZBTB8A on related signal transduction pathways. Dual-luciferase reporter assay system was performed toinvestigate the potential role of ZBTB8A on cell signaling pathways. The data indicatesthat ZBTB8A protein suppresses the activation of multiple cell signaling pathwaystranscription, especially on the pCRE-Luc, which has significant inhibitory effect andshows a dose-dependent. These results suggest that ZBTB8A protein may act as atranscription repressor in the activation of multiple cell signal transduction pathways tomediate cellular functions, thus affecting important physiological processes on cellproliferation, differentiation, migration, apoptosis and cell cycle.For expression of the recombinant protein, plasmid pGEX-4T-1-ZBTB8A wastransformed into competent BL21cells. When BL21cells harbouring apGEX-4T-1-ZBTB8A vector were grown to OD600of0.5-0.6, then IPTG was added to afinal concentration of0.6mM. The cells lyse was examined by SDS-PAGE andpotassium chloride staining to verify the localization of the expressed recombinantprotein. The purified ZBTB8A protein was obtained by electroeluting and dialyzing thebroken gels containing the recombinant GST-ZBTB8A protein. With anti-GST tagantibody, Western blot assay further verified the purified protein for the purpose ofprotein. BCA method determined of its concentration of1.254mg/mL. The rabbit wasinjected repeatedly with highly purified recombinant ZBTB8A protein. The antiserumagainst recombinant ZBTB8A was harvested from the carotid artery finally.The specificity of the antiserum was determined by western blot analysis. Theresults show that the produced polyclonal antibody against the purified recombinantZBTB8A protein exhibits high specificity, both BTB domain and ZNF domain ofZBTB8A are antigenic determinant—polyclonal antibody including against both itsBTB domain and ZNF domain. The polyclonal antibody can be used for the detection ofthe endogenous expression of ZBTB8A protein. Using agarose double-diffusionexperiment was to determine anti titer of1:32. With the ProteinG, the polyclonalantibody was purified, and then detected the purification effect by SDS-PAGE, thusobtaining relatively pure antibodies. All of these provide some support for furtherresearch.