Cloning and expression analysis of two hypothetical dense granule protein genes of Eimeria tenella in chickens
Eimeria tenella is a highly pathogenic protozoan that seriously endangers the healthy development of the chicken industry.Dense granule proteins(GRAs)are important intracellular parasitic functional proteins of the api-complexan protozoa,which have high immunological application value.However,there is no exact report on the dense granule proteins of coccidia.In order to explore the GRAs of Eimeria tenella and study their functions,two hy-pothetical dense granule proteins(hEtGRA),hEtGRA12 and hEtGRA9,were identified from E.tenella Beijing strain by bioinformatics,molecular biology and immunology.The purified proteins were used to immunize mice to prepare polyclonal antibodies,and the antigenicity of the two proteins was detected by ELISA and Western blot.The results of molecular cloning showed that the coding regions of hEtGRA12 and hEtGRA9 genes were 1 188 bp and 1 110 bp in length,encoding 395 and 369 amino acids,respectively.The similarity of hEtGRA12 and hEtGRA9 with GRA12 and GRA9 of other apicomplexan protozoa was 28.8%-39.6%and 27.5%-29.5%,respectively.SDS-PAGE showed that the recombinant proteins rhEtGRA12 and rhEtGRA9 were 63.6 ku and 67.0 ku,respective-ly.The results of ELISA and Western blot showed that the recombinant proteins rhEtGRA12 and rhEtGRA9 could in-duce high antibody levels in mice,and could be recognized by chicken anti-E.tenella positive serum,indicating good antigenicity.In this study,two hypothetical dense granule protein genes hEtGRA12 and hEtGRA9of Eimeria were identified,and recombinant proteins rhEtGRA12 and rhEtGRA9 were obtained,which laid a foundation for the study of gene function and immune application of dense granule protein of Eimeria.
万方数据
维普
