Authors : Lihua Fan, Yujiao Zhu, Wanlong Tan, Pengjie Yang and Chengyu Hu
Abstract: The grass carp (Ctenopharyngodon idellus) PKZ full-length cDNA (GU299765) had been cloned and identified recently. Within its N-terminal part of the protein there are two Z-DNA binding domains called Zα1 (1~67aa) and Zα2 (81~152aa). Zα domain is unique to PKZ and distinguishes them from other translation Initiation Factor 2 alpha (eIF2α)-kinase. To obtain polyclonal antibody against CiPKZ Zα, the PKZ Zα gene was amplified by PCR from the template obtained in the earlier research and identified by DNA sequence analysis. Then, it was digested by BamHI, XhoI and ligated with pET-32a vector which was by the same treatment. Sequenced and blasted with the NCBI GenBank, the recombinant plasmid pET-32a-PKZ Zα was obtained. The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced by 1 mmol L-1 IPTG. Researchers obtained CiPKZ Zα polypeptide via E. coli prokaryotic expression and purified with Ni-NTA His-Bind Resin affinity chromatography. Rabbit Polyclonal Antibody (Pab) against CiPKZ was raised using the purified N-terminal fragment of CiPKZ containing its Zα1 and Zα2 domains. Western blot analysis showed that the antibody had high affinity and specificity and higher titer. Immunohistochemistry assay identified that expression of PKZ could be detected in liver tissue.
Lihua Fan, Yujiao Zhu, Wanlong Tan, Pengjie Yang and Chengyu Hu, 2012. Preparation and Application of Polyclonal Antibody against PKZ from Ctenopharyngodon idellus (CiPKZ). Journal of Animal and Veterinary Advances, 11: 3169-3174.