| Trichinella spp.,There were thirteen different taxa[1]have been identified since it was first discovered.According to whether the larvae form cysts in the host muscle cell recombination process,it is mainly divided into two clades-cysts and no cysts.Trichinella spiralis（T1）and Trichinella pseudospiralis（T4）,as the representative species of the two clades,differ from the cysts in the susceptibility of the host and the inflammatory response after infection.T4 is the only species that can infect mammals and birds at the same time,and the rest only parasitize spinal mammal hosts.The reason for this difference between insect species is likely to be due to different excretory-secretory products（ESPs）.ESP is directly exposed to the host’s immune system and can directly act on the body’s cells to participate in regulation,thereby achieving long-term parasitism in the host.Its composition is complex and is closely related to the parasitic life and pathogenicity of the parasite.ESP has significant preventive and therapeutic effects on autoimmune diseases[2],but the direct parasitic Trichinella spiralis causes patients with psychological discomfort and pathological damage.In addition,the trichinella spiralis-derived protein composition is complex,and the concentration is not easy to control.Research and applications are limited.Then identification and screening of effective protein molecules in Trichinella spiralis can avoid the harm caused by Trichinella infection,and can replace Trichinella parasites to prevent Trichinella and treat autoimmune diseases and provide an experimental basis.This experiment is expected to start with T1,T4 excretion secretions,and preliminary screening at the protein level for related proteins that affect cyst formation and/or participate in immune regulation.Firstly,the excretory secretions of T1 and T4muscle larvae were collected.After SDS-PAGE identification of protein quality,iTRAQ technology（isobaric tags for relative and absolute quantitation）was used to identify the proteins and screen differentially expressed proteins.A total of 1027proteins were identified during this process,with 163 up-regulated proteins in T1 and31 up-regulated proteins in T4.The higher expression abundances in T1 are the DNaseⅡfamily（including Plancitoxin-1 with DNaseⅡactivity）,serine proteases and inhibitors,cystatin-like proteins,enolase,heat shock proteins,and nucleotide enzymes;T4 up-regulated expression mainly includes cadherin,DNaseⅡ,Moesin/ezrin/radixin protein,β-hexosaminidase,histone H2B/H4 and so on.In order to verify the accuracy of the results of iTRAQ experiments,real-time quantitative PCR was used to compare the transcription levels of selected genes encoding nine proteins.The results are consistent with protein levels,confirming the reliability of iTRAQ results.Subsequently,Trichinella cystatin-like protein was selected for subsequent preliminary exploration.A recombinant Trichinella cystatin-like protein-pET22b expression vector was successfully constructed based on the pET22b plasmid,and E.coli was used to express the recombinant Trichinella cystatin-like protein.Bioinformatics analysis showed that the Trichinella cystatin-like protein consists of 237 amino acids,a protein mass of 27.6 kDa,a theoretical isoelectric point of 8.02,and a signal peptide with an amino acid at the N-terminus of 18.The protein cell sublocalization shows that it is in the cytoplasm.Has 5 casein kinase II phosphorylation sites;3 N-myristoylation sites;3protein kinase C phosphorylation sites;and 1 tyrosine kinase phosphorylation site.There is no conserved cysteine protease inhibitor domain,and it has very low homology with cystatin of several existing parasitic nematodes.The protein may be a novel secretory cystatin-like protein from Trichinella spiralis.Anti-serum was obtained by immunizing rabbits with recombinant Trichinella cystatin-like protein,and the indirect immunofluorescence experiment of the anti-serum on the worm was found to be localized on the surface and cysts of the worm.Finally,mice were immunized with the recombinant cystatin-like protein,and after three immunizations,the deworming rate was calculated to investigate the protective effect of the cystatin-like protein on mice.The deworming rate showed that the average number of muscle larvae detected per mouse in the cystatin-like protein immunized group was significantly different from that in the PBS and adjuvant control groups after infection with T1 and T4（P<0.05）.T1 muscle larvae The worm reduction rate reached 60.54%,and the T4 muscle larvae reduction rate reached 37.49%.Recombinant cystatin-like protein of Trichinella spiralis has a good mitigation effect on T1 infected mice.In summary,on the one hand,this study conducted an overall analysis of the differential proteins excreted in the muscle larvae of T1 and T4,on the other hand,found a new type of recombinant spinal located on the body and cyst of Trichinella spiralis.The cystatin-like protein has a good worm-reducing effect on mice infected with T1 and T4. |
PDF Full Text Request