Expression And Subcelular Localization Of PKZ From Grass Carp(Ctenopharyngodon Idella) And Study On Histology And Morphology Of Two Kinds Of Fish

PKZ(protein kinase containing Z-DNA binding domain) is a kind of unique protein kinase newly found in fish,which belongs to not only the family of Z-DNA binding domain proteins but also the translation initiation factor2alpha (eIF2a)-kinase. The N-terminal part of the protein is regulatory domain..consists of Zal and Za2two parts of Z-DNA binding domains instead of dsRN A motifs.The C-terminal part of the protein is functionally distinct translation initiation factor2alpha (eIF2a)-kinase domain. Previous studies indicated that PKZ in fish transcript levels are known to be upregulated by IFN, the synthetic dsRNA polyI:C, viral infection and heat-shock etc.So PKZ in fish shares the similar feature with that of mammalian PKR, the results support a role for PKZ, like PKR, in host defense against virus infection,but also act as an adaptor in cells to response to many stimulis.DrPKZ could strongly down-regulated luciferase activity in both HEK293T and CHO cell lines which were cotransfected with the luciferase reporter plasmid.The investigation showed that Wild-type AsPKZ could strongly inhibited β-gal expression. Our earlier study has shown that PKZ could respond to the stress in cell, it inhibited protein synthesis and made cell apoptosis finally by phosphorylated eIF2a. PKZ in fish shares the typical feature of the translation initiation factor2alpha (eIF2a)-kinase.However, the exact role of CiPKZ in the"Stress/Z-DNA/Z-RNA/PKZ/eIF2a/apoptosis" pathway still remains unclear. Identification of the precise location of PKZ within the cell may provide some clues to its role as a regulator of the antiviral state,as well as of cell apoptosis and other processes.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 Zal (1-67aa) and Za2(81～152aa). Za domain is unique to PKZ and distinguishes them from other translation initiation factor2alpha (eIF2a)-kinase. Identification of the precise location of PKZ within the cell may provide some clues to its role as a regulator of the antiviral state and other processes.To obtain polyclonal antibody against CiPKZ Za, the PKZ Za gene was amplified by PCR from the template obtained in our previous work 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 Za was obtained.The recombinant plasmid was transformed into E. coli BL21(DE3) and induced by1mmol/L IPTG.We obtained CiPKZ Za 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 Zal and Za2domains. 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, kidney tissue and spleen tissue. CiPKZ is mainly exsisted in the cytoplasm,but some part of PKZ occures in the nucleolus of liver cells after induction by polyⅠ:C.Using RT-PCR to detect the specific expression in tissues of liver,spleen,kidney, intestine,heart and gill,the results indicated the PKZ is experessed in all tested tissues, and expressed to varying extents relative to the induction hour. PKZ protein is ubiquitously distributed in all examined tissues the CiPKZ.After induced by PolyⅠ:C for0h,6h,12h,18h,24h,48h,and induced by GCHV for0h,6h,12h,18h,24h,48h72h,respectly.The profile of PKZ were upregulated.Grass carp and carnivorous mullet intestinal histology was observed with light microscopy. The results showed that the grass carp intestinal wall could be divided into mucosa, submucosa,muscularis and serosa from the inner to the outer. Mucosa is made up of the epithelium, lamina propria and musculairs mucosa and so on. The intestinal histological structure characteristics of the carnivorous mullet are almost the same as that of grass carp. The differences are:mullet intestinal lumen is mini in diameter. Mucosal fold is longitudinal fold shaped, linear and more closely aligned. It is generally higher, having fewer and larger goblet cell and obvious central lacteal. Grass carp intestinal lumen is major in diameter. Mucosal fold is cross fold shaped, loosely aligned. It is generally lower, having more but smaller goblet cells and less obvious central lacteal.