Interaction Of Eimenria Tenella Rhomboid And Microneme Protein

With the development of modern poultry industry, coccidiosis has become a season of multiple disease. Prevention and treatment of coccidiosis has become the focus of research. Due to long-term use of traditional drug, drug resistant has emerged in coccidian. It is more difficult to control coccidiosis combined with the problem of drug residues. Immunization is available means of controlling the disease, but because of complexed life cycle and uncleared intrusive mechanism of the coccidia, the current vaccine was less effective. Choice of candidate genes for coccidiosis vaccines must be resolved as soon as possible. Microneme protein, an adhesion protein, is secreted by the microneme at the top of the sporozoite. MIC is a key protein which is used to recognize and bind the host cell receptors. Rhomboid protein has been attracted the researchers note all the time since it was discovered. As a class of intramembrane serine proteases, Rhomboid participate in the epidermal growth factor and epidermal growth factor receptor signal transduction. In recent years, many rhomboid proteins had also been found in some apicomplexan protozoa, including Eimeria tenella. After identification of rhomboid in Toxoplasma and Plasmodium, the result indicated that the proteolysis of rhomboid and MIC was a key process of host cell invasion. There is no reporte about E. tenella rhomboid protein function now. Whether MIC protein is hydrolyzed by the rhomboid, Whether the hydrolysis process is the key to the process of coccidia invasion, which are need to study.As an immune promoter, Yeast had already been studied and attention. A large number of glucan and mannan oligosaccharides are contained in yeast cells. Yeast can improve immunity, increase in antibody titer and the number of peripheral blood lymphocytes, thereby increasing the body's anti-bacterial and anti-cancer ability. However, as the immune enhancer, application of yeast need to be further expanded. Yeast will have far-reaching research value when it used to control parasitic disease. 3,4-dichloroisocoumarin (DCI) is a very effective serine protease inhibitor. Majority of serine protease sctivity can be losed by DCI. According to reports, Drosophila Rhomboid-1's activity could be inhibited by DCI and the EGFR signal transduction was influenced. Activity of Toxoplasma gondii TgROM5 could also be impacted. So the host cells invasion was inhibited. But there is no report has been published on DCI used to inhibit Eimeria tenella Rhomboid and against coccidiosis.In this study, first, suitability of E. tenella rhomboid for CytoTrap yeast two-hybrid system was detected. Then according to the Gal4 yeast two-hybrid system, bait plasmid pGBKT7-Rho, Capture plasmids pGADT7-MIC1 and pGADT7-MIC4 was successfully constructed. Plasmids were cotransformed into yeast cells AH109, respectively. E.tenella MIC4 was screened for interacted with Rhomboid protein. Then the interaction was further validated by coimmunoprecipitation. The yeast host cells which contained interacted protein was broken to immune chickens. Experiment of attacking E. tenella showed that chicken had got protective immunity. Anticoccidial index could reach 168.24. Finally, inhibited effect of sporozoites cell invasion was detected by DCI. It is the basis for molecular mechanism research of E. tenella invasion. It is also theoretical evidence for prevention and treatment of coccidiosis.Sequence analysis and verification of E. tenella rhomboid bait plasmid suitability for CytoTrap yeast two-hybrid system The rhomboid gene segment of E. tenella was generated and analyzed by RT-PCR with primers. According to the CytoTrap yeast two-hybrid system, rhomboid gene was subcloned into the plasmid pSos. Then bait and control plasmids were cotransformed into yeast cells cdc25H. Self-activation of Rhomboid was verified. The results showed that E. tenella Rhomboid gene open reading frame was successfully amplified. Recombinant expression vector bait pSos-Rho was constructed. Compared with other amino acid sequence of rhomboid-like protein, we supposed that EtROMz is EtROM3 in E. tenella. Western blot showed that the bait protein in yeast cells could be successfully expressed. Rhomboid was located in the vicinity of the host cell membrane of yeast cdc25H by indirect immunofluorescence. When bait and control plasmids were cotransformed into cells, ras pathway was activated and yeast cells grew on galactose medium at 37 ℃. That mean bait protein could self-activation. Thus rhomboid can not use the system to screen interacted protein.Screen interacted MIC proteins with the rhomboid First E. tenella MIC1 and MIC4 gene segment were generated. Then bait plasmid pGBKT7-ROM, capture plasmid pGADT7-MIC1 and pGADT7-MIC4 were constructed. Bait and capture plasmid were cotransformed into yeast cells AH109, respectively. At last, protein interaction could be identified by X-Gal verification. The results showed that the recombinant bait plasmid and capture plasmids were constructed successfully. The target gene could be expressed in yeast cells. There was no self-activation and toxicity for yeast. Interaction of rhomboid protein and MIC4 protein was identified by Gal4 yeast two-hybrid system. Western blot showed that MIC4 protein became more smaller. We presumed that it was due to the cutting action of rhomboid proteins.Interacted verification of rhomboid and MIC4 protein First, recombinant prokaryotic expression plasmid pET28a-MIC4, recombinant eukaryotic expression plasmid pcDNA-Myc-ROM and pcDNA-HA-MIC4 were constructed. Then expression, purification and renaturation of E. tenella MIC4 were performed. Interaction of rhomboid and MIC4 was verified by western blot. The recombinant eukaryotic expression plasmids were cotransfected HeLa cells and then interaction of rhomboid and MIC4 protein was verified by immunoprecipitation. The results showed that prokaryotic and eukaryotic expression plasmids were successfully constructed. The prokaryotic expressed rhomboid protein mixed with MIC4. Immunoprecipitation showed that there was interaction between two proteins. The eukaryotic recombinant plasmids were transfected HeLa cells. Indirect immunofluorescence showed rhomboid and MIC4 protein could be successfully expressed in HeLa cells. Immunoprecipitation and western blot proved that rhomboid interacted with MIC4 protein and enzyme activity of Rhomboid protein could cut their substrate MIC4 protein. The test result provides a theoretical basi for further study of the molecular mechanisms of coccidia invasion. Protective immunity of interacted protein in yeast against E.tenella challenge Yeast cells AH109 that contained rhomboid, MIC4 and interacted protein were broken. Chickens were immunized with recombinant proteins via subcutaneous route. Chickens were then challenged with E.tenella sporulated oocysts and the efficacy of immunization was evaluated on the basis of oocyst output, cecal lesion scores, ACI, response immunity of cellular and antibody. All the recombinant proteins immunized chickens developed specific immune responses, and there was a significant increases of the percentages of CD4+and CD8+cells and antibody titre compared to the control(P<0.05), especially interacted protein group (P<0.01). ACI of that group could reached 168.24. The yeast group also produced a good immune protection for coccidiosis. This resule was a theoretical basis of coccidiosis vaccine research.Inhibited effect of sporozoites cell invasion was detected by DCI. E. tenella sporozoites was cultured in vitro in primary chicken kidney cells. DCI was divided into two groups. One, sporozoites were dealed with DCI, and then add into the cells. Other, DCI and sporozoites were add into the cells together. The number of the first-generation schizonts, second-generation schizonts and progeny oocysts were counted. Inhibited effect of different DCI concentrations was identified. Results showed that chicken kidney cells invasion was inhibited by 20μM DCI. At the same time, the number of progeny coccidia oocysts was controled. DCI existed potential role of treatment coccidiosis and it will be become one candidate component for the anticoccidial drugs study.