The Preliminary Research On Two Conserved Proteins Of Eimeria Tenella

Coccidiosis has a high impact in the development of the poultry industry and cost great financial losses around the world. The disease is caused by several kinds of Eimeria spp which live in the chicken intestine. Among these kinds of Eimeria spp, Eimeria tenella is one of the most virulent parasite and mainly in chorionic epithelium and caecum. It can caused hemorrhagic enteritis which lead to death in severe infections. The Eimeria belongs to the Apicomplexa phylum which are obligate intracellular protozoa. Though different apicomplexan parasites specifically invade different cells, the cell invasion mechanism is highly conserved. When the parasite begin to adhere and invade to the host cell, a ring-shaped moving junction（MJ） is formed between the anterior end of the parasite and the host cell plasma membrane. Recent researches have indicated that the apical membrance protein-1（AMA1） which is secreted by micronemes is the key component of MJ, so the proteins that interact with AMA1 may participate in the formation of MJ and are essential during the parasite invasion the host cells. In orde to find some proteins that interacted with Et AMA1 and research the role of these proteins during the invasion process, Et AMA1 was selected as bait, ESTs encoded interaction proteins with Et AMA1 were obtained by screened from the yeast two hybrid c DNA library of E.tenella sporozoites by using bait. In this study we selected two ESTs（ No: 559 and 39）, then cloned and expressed. We also analysed the function of the proteins and verified the interaction relationship with Et AMA1.1. Gene cloning and bioinformatics analysis of Eimeria tenella CHP559 and CHP39The two ESTs（No.559 and No.39） both have a poly（A） in the 3’end, so the fragments of 5’ends of c DNA for the two genes were obtained by the rapid amplification of c DNA ends（RACE） technique. Two full-length c DNAs were cloned and sequenced based on the two ESTs sequence. A BLAST search of the E. tenella genome database found that the ORF sequences of two genes had 100% identity with ETH₀0024035 and ETH₀0029745, respectively, which encoded conserved hypothetical protein, so the two genes were designated Et CHP559 and Et CHP39, respectively. The full-length sequence c DNA of Et CHP559 was 1746 bp which included an ORF of 1224 bp encoding 407 amino acids with the predicted molecular weight of 46.04 k Da. The deduced amino acid sequence had a predicted transmembrane region, a signal peptide and 15 antigenic sites, but there was no conserved domain. The full-length sequence c DNA of Et CHP39 was 1344 bp which included an ORF of 873 bp encoding 290 amino acids with the predicted molecular weight of 32.6 k Da. The deduced amino acid sequence had no predicted transmembrane region, no signal peptide and no conserved domain, but had 9 antigenic sites. Expression profiles of Et CHP559 and Et CHP39 in E.tenella unsporulated oocysts, sporulated oocysts, sporozoites and second-generation merozoites were determined using real-time quantitative PCR. Among these four development stages, the m RNA level of Et CHP559 was higher in sporulated oocysts and sporozoites than other two stages, moreover, the transcript level was highest in sporozoites and lowest in second generation merozoites. The m RNA level of Et CHP39 were higher in sporulated oocysts and unsporulated oocysts than sporozoites and second-generation merozoites. Moreover, the transcript level was highest in sporulated oocysts and lowest inecond-generation merozoites. These results showed the two genes were differential expressed in different development stages.2. Prokaryotic expression and preliminary study of Et CHP559 and Et CHP39 genetic characteristicsThe amplified Et CHP559 gene was cloned into p GEX-4T-2 vector and p Cold-TF vector, respectively. And the recombinant plasmids were transformed into competent cells E.coli BL21, then induced to express recombinant protein GST-Et CHP559 and His-Et CHP559 by IPTG induction. The GST-Et CHP559 protein was expressed as inclusion bodies and the His-Et CHP559 protein was soluble expressed. The amplified Et CHP39 gene was cloned into p ET28 a vector, and induced to express recombinant protein His-Et CHP39 as inclusion bodies. The recombinant protein His-Et CHP559 and His-Et CHP559 were purified by the Ni-column affinity chromatography and cutting the gel slices, respectively. The polyclonal antibodies were obtained by immuning rabbits with purified recombinant proteins. Western-blot revealed that the recombinant proteins His-Et CHP559 and His-Et CHP559 all had excellent reactionogenicity and immunogenicity. Using the polyclonal antibody, the dispersed localision of the native proteins Et CHP559 and Et CHP39 during the process of invasion and development of sporozoites in DF-1 cells were investigated by immunofluorescence, respectively. The Et CHP559 protein located on the parasite surface before invasion, concentrated to the anterior of sporozoite with the increase of protein expression when the sporozoites begin to invade the DF-1 cells. It mainly located on the anterior end after the sporozoites added into DF-1 cells for 2 h, and was secreted to the tail end with the increase of protein expression after sporozoites added into DF-1 cells for 24 h. The Et CHP39 protein located on the anterior end of sporozoite before invasion, was secreted to the surface and the anterior end when the sporozoites begin to invade, it was also found at the anterior end after infection for 2 h, then moved to the tail end with the increase of protein expression after infection for 24 h, It also located on the anterior end of merozoite. To study Et CHP559 and Et CHP39 proteins in E. tenalla sporozoite invasion of DF-1 cells, invasion inhibition assays were performed by blocking sporozoites by preincubation with r Et CHP559 and r Et CHP39 antibody before DF-1 cell infection. Compared with the same dose of naive rabbit Ig G used as a negative control, the anti-r Et CHP559 Ig G significantly decreased invasion. Inhibition rate was as high as 70% at 300 μg.m L-1 anti-r Et CHP559 Ig G, while na?ve rabbit sera Ig G did not have a significant effect on invasion. Compared with the control group, the invade capacity of anti-r Et CHP39 antibodies group was decreased, and the inhibition rate was 30% at the anti-r Et CHP39 Ig G concentration of 400 μg.m L-1.3. Verification of the interaction relationship of the Et CHP559 and Et CHP39 proteins with the Et AMA1 proteinIn this study, the eukaryotic expression protein was used to verify the interaction with Et AMA1. Firstly, the six eukaryotic expression recombinant plasmids were constructed including pc DNA3.1-flag-Et CHP559, pc DNA3.1-flag-Et CHP39, pc DNA3.1-Et AMA1, p Bi FC-VC155-Et CHP559, p Bi FC-VC155-Et CHP39 and p Bi FC-VN155-Et AMA1. The recombinant plasmids, after identified with PCR and enzymes digestion, were transfected into DF-1 cells. The expression proteins in the transfected DF-1cells were successfully confirmed by western blot and indirect immunofluorescence assay. After the DF-1 cells were transformed with recombinant plasmids pc DNA3.1-flag-Et CHP559, pc DNA3.1-flag-Et CHP39 and pc DNA3.1-Et AMA1 for 48 h, the CO-IP assays was performed. And the results showed that the monoclonal antibody of flag can precipitate the Et CHP559 and Et CHP39 protein, but not the Et AMA1 protein at the same time. After the DF-1 cells were transformed with recombinant plasmids p Bi FC-VC155-Et CHP559, p Bi FC-VC155-Et CHP39, p Bi FC-VN155-Et AMA1 for 30 h, the bimolecular fluorescence complementation assays were performed. And the results showed that green fluorescence was not detected in the DF-1cells transformed with the experimental recombinant plasmids and negative plasmids, while green fluorescence was observed in the DF-1cells transformed with the positive control plasmids It indicated that there maybe not interaction relationship of the Et CHP559 and Et CHP39 protein with the Et AMA1 protein...