| Haemonchus contortus is a parasitic nematode that parasitizes in the abomasum of ruminants such as cattle and sheep.They live on the blood of the host,causing host anemia and digestive system syndrome,then seriously affecting the health of the host animal.At present,H.contortus has become one of the biggest threats in cattle and sheep production worldwide.The prevention and control method mainly relies on the use of anthelmintic drugs.However,recent years,drug resistance and residual effects are incredibly increasing.Therefore,there is an urgent need to develop new drugs and vaccines to control the disease.It is known that H.contortus has diapause phenomenon,although the relevant mechanism is not clear now.According to the research,it is speculated that the diapause mechanism of H.contortus is similar to the C.elegans.The pheromone that triggers the diapause phenomenon of C elegans is produced by the peroxisome β-oxidation reaction pathway.Four kinds of enzymes are involved in this process,namely acyl-CoA oxidase(ACOX1)and enoyl-CoA hydratase(MAOC1),hydroxyalkane-CoA dehydrogenase(DHS28)and 3-ketoacyl-CoA thiolase(DAF22).These peroxisome metabolizing enzymes have a vital impact on the growth and development of C.elegans.This experiment intends to study the related functions of Haemonchus contortus 3-ketoacyl-CoA thiolase(Hc-DAF22),mainly in the following aspects.1.It is clear that Hc-DAF22 is localized in peroxisomes with PTS1 and PTS2In order to study the function of Hc-DAF22 protein,we plan to explore its subcellular location.We use the uniprot website to predict the subcellular localization and localization signal of Hc-DAF22 protein,and use the fusion reporter protein? localization method to verify the prediction results.The prediction results show that Hc-DAF22 protein is targeted to peroxisome with peroxisome targeting signal 1 and peroxisome targeting signal 2.The fusion gene expression vector pEGFP-C2-Hc-daf22 was transfected into HEK 293 T cells.The results showed that the Hc-DAF22 protein aggregated and distributed in the cytoplasm and was co-existed with the peroxisome marker plasmid(C2-m Cherry-pox),but there is no co-localization with the mitochondrial marker plasmid(C2-m Cherry-mito).pEGFP-C2-Hc-daf22-ΔPTS1,pEGFP-C2-Hc-daf22-ΔPTS2 and pEGFP-C2-Hc-daf22-ΔPTS1+2were removed the PTS1 and PTS2 and both of them.Then we transfect the above three vectors into HEK 293 T cells,and the results show that removing PTS1 or PTS2 alone,the protein can be accurately located in the peroxidase.Only when PTS1 and PTS2 are removed at the same time,the punctate aggregation of the protein in the cytoplasm disappeared,showing a diffuse distribution in the cytoplasm,it means that it could not be accurately located in the peroxisome.In summary,Hc-DAF22 protein is localized in the subcellular organelle peroxisome,and has both PTS1 and PTS2.It provides a realistic basis for clarifying that Hc-DAF22 protein participates in the peroxisome β oxidation reaction in Haemonchus contortus.2.Study on the interaction between Hc-DAF22 protein and peroxisome transport protein Hc-PEX5In order to have a deeper understanding of the function of Hc-DAF22 protein and its molecular mechanism,the interaction protein of Hc-DAF22 was predicted and screened.On the STRING website,it is predicted that Ce-DAF22(a highly homologous protein of Hc-DAF22)has 11 interacting proteins.And two proteins related to peroxisomes,Ce-PRX5 and Ce-LONP2,are selected.The homologous sequences of H.contortus were compared in the NCBI database,then Hc-pex5 and Hc-lonp2 sequences were amplified in Haemonchus contortus.The eukaryotic expression plasmid p CDNA3.1-Hc-daf22-Flag,p CDNA3.1-Hc-pex5-HA,p CDNA3.1-Hc-lonp2-HA was constructed.Then,we use Co-IP method to explore whether Hc-PEX5 and Hc-LONP2 protein interact with Hc-DAF22 protein,and verify through GST-pull down.The results show that there is an interaction between the Hc-DAF22 protein and the Hc-PEX5 protein,but there is no interaction between the Hc-DAF22 protein and the Hc-LONP2 protein.We Constructed a eukaryotic expression vector that removes the peroxisome localization signal,and then used Co-IP and yeast two-hybrid experiments to explore whether PTS1 and PTS2 are the key sites for the interaction of Hc-DAF22 and Hc-PEX5 proteins.The results show that PTS1 is the key site of Hc-DAF22 and Hc-PEX5 protein interaction,while PTS2 is not the key site of Hc-DAF22 and Hc-PEX5 protein interaction.In summary,there is an interaction between Hc-DAF22 and the peroxisome transport protein Hc-PEX5,and PTS1 is the key site for the interaction between them.It laid the foundation for further research on the molecular mechanism of Hc-DAF22 protein importing into peroxisomes.3.PEX5 can mediate the import of Hc-DAF22 protein into peroxisomesIn order to study the mechanism of the import of Hc-daf22 protein into peroxisomes,the overexpression vector of Hc-daf22 was constructed,and Hc-daf22 was overexpressed in the model organism C.elegans by microinjection.The interference vector L4440-Ce-prx5 was constructed.We Used the feeding method to perform RNAi experiments on this overexpression strain,and observed the effect on the import of Hc-daf22 protein into peroxisomes.The results show that Hc-daf22 is overexpressed in Caenorhabditis elegans,and its protein presents obvious point-like aggregation distribution in the intestine,subcutaneous and other parts.After the interference of Ce-prx5,the Hc-daf22 protein cannot be imported into peroxisome,indicating that the peroxisome transport protein Ce-PRX5 can mediate the transport of Hc-daf22 protein.In order to further clarify the effect of Hc-pex5 on the function of Hc-daf22,RNA interference experiments were performed on H.contortus.The interference vectors L4440-Hc-pex5 and L4440-Hc-daf22 were constructed,and the feeding method was used to interfere with eggs of H.contortus.The changes of gene transcription levels and the development of worms were detected on the 1,3,and 7 days after RNAi.The experimental results showed that the transcription level of Hc-daf22 was significantly down-regulated at the L3 stage;compared with the control group,the Hc-daf22 interference group was significantly slower.Hc-pex5 and Hc-daf22 double gene interference group,compared with Hc-daf22 single gene interference group,development delay is more obvious.It shows that in H.contortus,Hc-pex5 has an effect on the function of Hc-daf22,which provides a practical basis for the study of the mechanism by which Hc-pex5 can mediate the import of Hc-daf22 protein into peroxisomes.In addition,the experimental results also show that the Hc-daf22 gene and Hc-pex5 gene play an important regulatory role in the growth and development of H.contortus,which provides effective data for the search and development of vaccine targets for H.contortus.In summary,we explored the subcellular localization of Hc-daf22 through the eukaryotic expression system,predicted and verified the effect of peroxisome targeting signal 1(PTS1)and peroxisome targeting signal 2(PTS2)on the subcellular localization of Hc-daf22.Through the STRING interaction protein prediction website,the proteins that interact with Hc-daf22 were predicted and screened,using Co-IP,GST-pull down and yeast two-hybrid experiments we verified the interaction between Hc-daf22 and Hc-pex5;The mechanism of the import of Hc-daf22 into peroxisomes was preliminarily explored.It was proved in C.elegans that Ce-PRX5 can mediate the import of Hc-daf22 protein into peroxisomes.In H.contortus,Hc-pex5 may mediate the import of Hc-daf22 protein into peroxisomes.In addition,it was discovered that Hc-daf22 gene and Hc-pex5 gene play an important role in the growth and development of H.contortus.It builds a foundation for studying the relevant molecular mechanism of Hc-daf22 in H.contortus peroxide β oxidation reaction and the development of new drugs and vaccines for H.contortus. |
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