Cloning,Expression and Preliminary Analysis of the Characteristics of the Tubulin Beta Chain Gene EtTBC of Eimeria tenella
Coccidiosis is an intestinal disease caused by parasites of the genus Eimeria spp.and one of the most important diseases affecting the poultry industry.At present,the control of coccidiosis mainly depends on the use of anticoccidial drugs,but drug resistance of Eimeria develops qucikly due to the long-term and extensive use of drugs.In order to study drug resistant mechanism of Eimeria,we previous study transcriptome sequencing on drug-sensitive strain(DS),diclazuril-resistant strain(DZR),maduramycin-resistant strain(MRR),salinomycin-resistant strain(SMR)of E.tenella.We found that the tubulin bate chain(Et TBC)was highly expressed only in DZR.Based on this,the EtTBC gene was cloned and expressed in this study,and its function was preliminarily analyzed.EtTBC was successfully cloned,the recombinant plasmid pET28a-EtTBC was constructed and the recombinant protein(rEtTBC)was successfully expressed.The mRNA transcription levels of E.tenella at different developmental stages of DS and in different strains(drug-sensitive strain,drug-resistant strains and field isolated diclazuril-resistant strains)were analyzed by qPCR.The results showed that the mRNA transcript level of this gene was highest in the unsporulated oocyst stage,while the other three stages were low in E.tenella DS;Moreover,its mRNA levels was only upregulated in DZR.Indirect immunofluorescence localization showed that the protein was mainly distributed on the cytoplasm and surface of sporozoites and second generation merozoites.Therefore,it is speculated that this protein may be involved in the invasion of the host and the immune evasion and the development of resistance of E.tenella to diclazuril.
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