The Primary Research Of Calcium Dependent Protein Kinase 4 From Eimeria Tenella

In order to understand the mechanism of invasion and development of Eimeria spp. EtCDPK4 genes of Eimeria tenella were studied. In this paper, the genetic characteristics and protein function of EtCDPK4 were explored.1. Gene cloning and Bioinformatics analysis of E. tenella Calcium Dependent Protein Kinase 4The full-length 5’-end and 3’-end of the EtCDPK4 cDNA were obtained by rapid amplification of the cDNA ends(RACE) with the GeneRacer? kit. The full-length cDNA of EtCDPK4 was 2,499 bp, including a single open reading frame(ORF) of 1,803 bp(positions 186 bp-1988 bp), a 5’-untranslated regions(UTR) of 185 bp and a 3’- UTR of 511 bp contained the characteristic poly(A) tail(AAAAAA) and no the classical polyadenylation signal(AATAAA). The ORF was deduced to encode a polypeptide of 600 amino acid residues with a calculated molecular mass of approximately 68.3 kDa. By TMPRED Program analysis of the transmembrane regions of the amino acid sequence of EtCDPK4, it is found that this protein has no transmembrane structure region. SignalP program analysis revealed that the protein had no signal peptide. The genetically mobile domain and conservative structure predicted that the protein contained distinct characteristic domains of the CDPK, including an amino-terminal kinase domain containing a typical serine/-threonine kinase active site, a carboxy-terminal calmodulin-like domain with 3 EF-hand motifs and an amino-terminal calmodulin-like domain with 2 EF-hand motifs for calcium-binding. To determine the mRNA transcription level of EtCDPK4 in different development stages(unsporulated oocysts, sporulated oocysts, sporozoites, the second-generation merozoites) of E. tenella, total RNA was extracted and subjected to qPCR analysis. Among 4 development stages, the mRNA level of EtCDPK4 was much higher in sporozoites than the other 3 stages, and the transcripts were hardly detectable in unsporulated oocysts and the second generation merozoites.2. Preliminary study for Calcium Dependent Protein Kinase 4 of Eimeria tenella protein expression and characteristicsThe amplified EtCDPK4 gene was cloned into pColdⅠvector. And the recombinant plasmids were transformed into competent cells E.coli BL21, then induced to express rEtCDPK4 by IPTG induction. rEtCDPK4 protein was expressed as soluble protein and purified by the Ni-column affinity chromatography. The polyclonal antibodies were obtained by immunized rabbits with purified recombinant proteins. Western-blot revealed that the rEtCDPK4 had excellent reactionogenicity and immunogenicity. Anti-rEtCDPK4 antibodies were applied to detect the localization of EtCDPK4 in sporozoites and during the first schizogony. EtCDPK4 exhibited a homogenous distribution pattern throughout the cytoplasm of sporozoites except the refractive body and the second-generation merozoites(Mrz) incubated in PBS for 2h. By contrast, when sporozoites were incubated in culture medium, EtCDPK4 expression had not changed significantly. After sporozoites invaded host cells, the localization of EtCDPK4 was mainly at the cytoplasm of the parasites except the refractive body for 12 h. Moreover, the green fluorescence intensity was enhanced at this phase. Foci of intense EtCDPK4 staining were also closely associated with the parasitophorous vacuole membrane(PVM). Observations 36 h p.i. showed that EtCDPK4 protein expression increased and was distributed in trophozoites. The labeled EtCDPK4 eventually became uniformly dispersed in immature and mature schizonts and decreased in immature schizonts. After the release of the first-generation merozoites from mature schizonts in DF-1, the labelling became very strong. To evaluate EtCDPK4 in the invasion of DF-1 cells by E. tenella sporozoites, an invasion inhibition assay was performed by blocking protein function by pre-incubation of sporozoites with antibody against rEtCDPK4 before DF-1 cell infection. The data showed that anti-rEtCDPK4 inhibited the invasion to 52 % at an antibody concentration of 400 μg/mL of the level seen with infection compared with non-treated sporozoites. The observed inhibition effect was dose dependent. By comparative analysis, the native rabbit-sera IgG did not have a significant effect on invasion process by E. tenella sporozoites. The presence of EtCDPK4 in unsporulated oocysts, sporulated oocysts, sporozoites and second-generation merozoites was determined by immunoblotting using antibodies obtained from rabbits immunized with rEtCDPK4. Western Blotting of the total proteins revealed that anti-rEtCDPK4 antibodies strongly labeled the same 68.3-kDa bands in second-generation merozoites, unsporulated oocysts and sporozoites but weaker reactivity in sporulated oocysts. The EtCDPK4 proteins probed with anti-rEtCDPK4 antibodies showed EtCDPK4 had high expression in second-generation merozoites, sporozoites and unsporulated oocysts, while little expression in sporulated oocysts.3. Preliminary study for kinase catalytic activity of Eimeria tenella Calcium Dependent Protein Kinase 4The proenzyme protein of r EtCDPK4 was purified by column affinity chromatography. Protein kinase activity of rEtCDPK4 was measured using a commercial kit based on the non-radioactive detection of animal PKC. The test relies on the in vitro phosphorylation of a PKC-specific fluorescent, synthetic peptide substrate. The resulting phospho-peptide was separated from the remaining non-phosphorylated peptides by agarose gel electrophoresis, detected under UV light, and quantified by means of a spectrophotometric assay. The reaction rate that the kinase activity of rEtCDPK4 was close to 0.87 nmol·min-1 ·ml-1 protein. The enzyme activity was found to be strongly dependent on Ca2+ concentrations with a calculated Ka value of 10 μM-50 μM, the proenzyme protein of rEtCDPK4 was incubated in the presence of various Ca2+ concentrations as indicated on the in vitro phosphorylation. The Ca2+ concentrations of 0, the rEtCDPK4 was completely inactivated so that the substrate could not be phosphorylated. With the increasing of Ca2+ concentrations, it can be seen that the activity of rEtCDPK4 was constantly enhanced in spite of the band relatively weak. The seven kinds of inhibitors were applied in the rEtCDPK4 activity assay by changing the phosphorylation reaction system and invasion inhibited assay. The results shown that W-7, H-7, H-89 and Myristoylated peptide were found to significantly decrease the sporozoites invasion activity.4. Preliminary screening the interacting proteins with Eimeria tenella Calcium Dependent Protein Kinase 4 by Yeast Two-HybridIn order to construct the bait plasmid applied in yeast two-hybrid system, the EtCDPK4 fragment of E. tenella was amplified and ligated to pGBKT7, the yeast expression vector, and the bait plasmid pGBKT7-EtCDPK4 was constructed. Transcriptional activation, toxicity and expression of bait in yeast cells were tested. These results indicated that the bait plasmid pGBKT7-EtCDPK4 was successfully constructed without activation and toxicity, the protein was expressed in yeast cells. The bait plasmid pGBKT7-EtCDPK4 could be applied in screening the yeast two-hybrid cDNA library from sporozoites or second generation merozoites of E. tenella. In order to screen the interacting proteins with EtCDPK4 in sporozoites or merozoites during invading the epithelial cells, the yeast two-hybrid cDNA library of sporozoites or second generation merozoites of E. tenella that constructed in our lab and the bait plasmid pGBKT7-EtCDPK4 were applied in yeast two-hybrid system, and the high selective medium SD/-Ade/-His/-Trp/-Leu/X-α-GAL was used to screen the interactiing proteins. The result showed that 88 blue clones were acquired and and transformed into E. coil TOP10 compentent cell. Finally, 80 positive plasmids were successfullyobtained and sequenced after PCR identification. 14 ESTs of positive plasmids were obtained, which had 4 ESTs sequence of E. tenella belonging to the whole genome. Based on the above analysis, 10 putative interacting proteins with EtCDPK4 were screened by yeast two-hybrid assay, including 9 unknown ESTs, 6 unnamed proteins and 4 reported proteins(EtSerpin, Etm094G10, Etm109G06, EtGMP synthase). The interaction between EtSerpin and EtCDPK4 was verified by using the Co-IP method. The result showed that EtCDPK4 interacted with EtSerpin in the sporozoites or second generation merozoites from E. tenella.