Expression of the POZ domain of human Zbtb7A and preparation of its polyclonal antibodies / 医学研究生学报

ABSTRACT

Objective: Proto-oncogene Zbtb7A has been characterized as a molecular switch in the process of cancer initiation and development.Our goal is to obtain the POZ domain of the Zbtb7A protein and prepare its polyclonal antibodies.Methods: We optimized the coding sequence of the POZ domain according to the codon bias of E.coli and synthesized the sequence with two-step PCR,which was then introduced into the pET-26b(+) vector to express the protein.The recombinant protein was analyzed by 15% SDS-PAGE and the corresponding band was cut out from gel.The minced gel slice that contained the POZ domain protein was injected to immunize rabbits.The collected rabbit antiserum was purified using the saturated ammonium sulfate method in combination with protein G antibody purification,and the purified polyclonal antibodies was evaluated by Western blot.Results: The optimized sequence of the POZ domain was correctly obtained and successfully constructed into the pET-26b(+) vector.After induction,an expected protein band about 14 KD was detected on 15% SDS-PAGE,and highly purified polyclonal antibodies were obtained,which were specifically bound to human Zbtb7A.Conclusion: The obtained recombinant protein of the POZ domain and its polyclonal antibodies can be used for further studies of Zbtb7A.