Characteristics And Function Prediction Of Eg TAK1 Protein And Its Role In Eg Development And Differentiation

Objective: 1)To predict the physicochemical and structural characteristics and biological functions of Eg TAK1 protein;2)To explore the regulatory effect of Eg TAK1 protein on the development and differentiation of Echinococcosis granulosus after bile acid stimulation.It provides a theoretical basis for screening target genes that inhibit the development and differentiation of Echinococcosis granulosus,which is helpful for early prevention and control of pathogen environmental pollution and cutting off host transmission.Methods: 1)Echinococcosis granulosus protoscoleces were collected from the liver of sheep naturally infected with Echinococcosis granulosus in a slaughterhouse in Urumqi,Xinjiang.The protoscoleces were cultured in with or without sodium taurocholate(T4009),and compared and observed the development and morphological changes of protoscoleces in different time periods.2)The full-length ORF of Eg TAK1 was amplified by PCR and sequenced.The physicochemical and structural characteristics and biological functions of Eg TAK1 protein were predicted by bioinformatics software.3)Based on the prediction of the characteristics and functions of the protein,the recombinant plasmid p ET30a-Eg TAK1 was constructed and expressed in the prokaryotic expression system.The purified recombinant protein was used to immunize New Zealand white rabbits to prepare polyclonal antibodies.The antibody titer was detected by ELISA,and the recognition of polyclonal antibodies to recombination Eg TAK1 protein was identified and verified by Western Blotting.4)The expression levels of Eg TAK1 in different stages of Echinococcosis granulosus development with and without T4009 were detected by realtime fluorescence quantitative PCR and Western blotting.Using RT-q PCR to compare the effects of different stages of dog bile culture and T4009 culture on the expression of Eg TAK1.The role of this protein in the key developmental stages of Echinococcosis granulosus was determined by immunohistochemistry.5)Three targeted si RNAs(Eg TAK1-si RNA1—Eg TAK1-si RNA3),a negative control si RNA(NC si RNA)and Cy3 fluorescence control were designed according to the cloned and sequenced Eg TAK1 ORF sequence.The five si RNAs were transfected into the newly collected protoscoleces of Eg by electroporation at a concentration of 5μmol/L,and the samples were collected after 3 days of culture.The interference effect of si RNA on the target gene was detected by RT-q PCR.Results: 1)Compared with the first day,PSC without T4009 culture showed a larger and elliptical capsule shape on the third day,and developed into a short and thick type after 2 weeks;PSC cultured with T4009 are in the T4009 stimulation stage on the 1st day,with an eversion rate of about 40% on the 3rd day,and an eversion rate of about 85% after 2 weeks.2)The fulllength ORF of Eg TAK1 gene was 1116 bp,and the encoded protein contained 371 amino acids.It was predicted that the protein was unstable and hydrophilic,and had good immunogenicity.It belonged to the protein kinase C-like(PKC-like)family,without transmembrane domain and signal peptide.The subcellular localization was located in the cell membrane,cytoplasm and nucleus.The secondary structure was mainly random coil.The sequence homology with the TAK1 of Echinococcus multilocularis was 83%,which was considered to be a highly conserved kinase;3)The recombinant protein showed a specific band with a molecular weight of about 50 KDa.The purified serum titer was 1 :128000,and the polyclonal antibody was successfully prepared.The polyclonal antibody could specifically recognize the immunogen and Eg TAK1 protein.4)During the development and differentiation of Echinococcosis granulosus,the expression of Eg TAK1 was the lowest in the germinal layer and the highest in the 35 d adult.The expression of Eg TAK1 in the germinal layer or adult stage was significantly different from that in the protoscoleces,the difference was statistically significant(P < 0.05).The expression of Eg TAK1 in T4009 group was higher than that in without T4009 group at 1d,3d and 17 d in vitro,the difference was statistically significant(P < 0.05).The expression of Eg TAK1 protein in T4009 group was higher than that in without T4009 group at 30 min,3h and 7d in vitro(P < 0.05).The effect of dog bile culture and T4009 culture on the development of Eg TAK1 m RNA in Echinococcosis granulosus were consistent,the different was not statistically significant(P > 0.05),indicating that sodium taurocholate play a major role in dog bile.Eg TAK1 protein was mainly expressed on the calcium particle envelope of PSC.In PSC without bile salt stimulation(3h),it was mainly expressed on the calcium particle envelope of the surface layer of the worm,while in the same period with bile salt stimulation,it was expressed on the calcium particle envelope of the scolex.In the 35 d adult stage,it is expressed in the head segments and immature segments of the worm,and also expressed on the calcium granule envelope;5)RNAi silencing of Eg TAK1 expression,the expression levels of Eg TAK1-si RNA1,Eg TAK1-si RNA2,Eg TAK1-si RNA3 transfection group and NC group were reduced by 76 %,42 %,19 % and 12 %,respectively,compared with untreated group(UT),si RNA1 decreased most significantly,the difference was statistically significant(P < 0.05).Conclusion: Conclusion : This study first predicted the physicochemical properties and biological functions of Eg TAK1 protein;preliminary verification of Eg TAK1 protein in Eg development and differentiation has a certain role in promoting.Eg TAK1 protein may be one of the intervention targets to inhibit Echinococcosis granulosus development,which can provide a theoretical basis for blocking the development and differentiation of Echinococcosis granulosus and cutting off host transmission.