Studies On Calcium-dependent Protein Kinases Of Eimeria Tenella

Eimeria species, causative agents of avian coccidiosis, are the most important parasites in poultry. They impair bird growth and feed utilization, cause increased mortality rates, and lead worldwide to tremendous economic losses in the poultry industry. So far, conventional disease control strategies have mainly relied on prophylactic medication and immunization with live vaccines. Because of diffusion of species, drug residues, and the emergence of drug resistant parasite etc, novel approaches are urgently needed to find to control coccidiosis.The life cycle of Eimeria is complex and consists of an exogenous phase and an endogenous phase. As different developmental stages have different morphological characteristics and living environments, the gene expression profiles differ significantly between the developmental stages and the functions of these genes are different. Sporozoite is the first important invasive stage to invade into the chicken intestine cells. In order to understand the mechanism of invasion and development of Eimeria, some differential expressed genes of sporozoites were studied. In this paper, two new differential expressed genes of sporozoites were cloned and expressed, then the functions were analysed.1 Cloning and analysis of two new genes from sporozoites of Eimeria tenellaBased on a lot of ESTs of differential expressed genes from sporozoites of Eimeria tenella by SSH and cDNA microarray in our laboratory, two ESTs (clone no.:ZB1-H07, ZB7-C11) which were up-regulated in sporozoites were selected to study. The Blast homology analysis of two genes showed that ZB1-H07 has high homology with calcium-dependent protein kinase (CDPK)of Plasmodium falciparum and ZB7-C11 has part homology with calcium-dependent protein kinase of Plasmodium chabaudi. The full cDNAs of two new genes were cloned with RACE. ZB1-H07 gene is 1 336 bp, containing a 1 302 bp of the ORF, encoding 433 amino acids with predicted molecular weight of protein 49.3 kDa. ZB7-C11 gene is 1 439 bp, containing a 525 bp of the ORF, encoding 174 amino acids with predicted molecular weight of protein 18.7 kDa. The property and function of proteins encoded by two differential expressed genes were predicted and analysized with bioinformatics analysis, including conserved functional domain of the encoded protetrans-membrane structure, antigen site, hydrophobic property, signal peptide, conserved domain etc. The protein encoded by ZB1-H07 has a high degree of homology with calcium-dependent protein kinase (CDPK) of Taxoplasma gondii, supposing the protein as calcium-dependent protein kinase of Eimeria tenella, named EtCDPK. The protein encoded by ZB7-C11 has only a small part homology with the protein of calcium-dependent protein kinase of Plasmodium chabaudi, suggesting the protein as a high expression new gene of sporozoites, tentatively named EtC11.2 The expression of EtCDPK and EtC11 gene and immunogenicity analysisEtCDPK gene was ligated to prokaryotic expression vector pET28a(+). The recombinant plasmids pET(28a)-EtCDPK was constructed and transformed into BL21(DE3) for expression. The recombinant protein (His-EtCDPK) could be highly expressed in the form of inclusion bodies in E. coli. The recombinant protein His-EtCDPK was purified using nickel affinity chromatography, and western-blot analysis showed the recombinant protein had good antigenic specificity and immunoreactivity. To obtain soluble protein and further analyse its function, EtCDPK gene was ligated to expression vector pPIC-9K and was transformed into GS115 pichia pastoris cells. The soluble protein obtained after induction with methanol also had good antigenic specificity and immunoreactivity by western-blot analysis. The soluble protein could be used to further study for its fuctions.EtC11 gene was ligated to prokaryotic expression vector pGEX-4T-2. The recombinant plasmids pGEX-4T-EtC11 was constructed and transformed into BL21(DE3) for expression. The recombinant protein GST-EtC11 could be highly expressed in soluble form in E. coli. The recombinant protein GST-EtC11 was purified using GST chromatography. Western-blot analysis showed that the recombinant protein had good antigenic specificity and immunoreactivity.3 Preliminary analysis of EtCDPK function 3.1 Immunofluorescence localization of EtCDPKIn order to investigate the dispersed localision of EtCDPK during the process of invasion and development of sporozoites, the sporozoites of Eimeria tenella were purified and added into chicken fibroblast cell (DF-1). The process of invasion and development of sporozoites in DF-1 cells within 48 h were observed by immunofluorescence microscopy. The sporozoites were incubated with PBS for 1 h, the pre-prepared anti-rabbit serum as first antibody, goat anti-rabbit fluorescent antibodies as secondary antibody. After sporozoites incubated in DF-1 for 2 h,EtCDPK was concentrated at the apical end of the sporozoites. With the development of sporozoites in DF-1,EtCDPK was detected in parasitolophorous vacuole (PV).3.2 Inhibition of Eimeria tenella invasionWith a view to evaluate the implication of EtCDPK in vasion of sporozoites of Eimeria tenella, the fresh sporozoites were labbled with CFSE for 1 h, then the labbled sporozoites were incubated with the pre-prepared anti-rabbit serum for 2 h, the sporozoites were added into DF-1 cell. After 20 h of culture, the cells were washed and analyzed by flow cytometry instrument. Data showed that anti-EtCDPK antibody was able to inhibitthe sporozoite invasion to a level of 68.8% compared with infected with non-treated sporozoites. This result suggested EtCDPK was involved with the invasion of the sporozoites and played an important role in the invasion of the sporozoites.