Expression On Spm-N Gene Of Theileria Annulata And The Development Of Indirect ELISA Method

In order to study the function of spm-N gene of the Theileria annulata and establish a diagnostic method for rapid detection of T.annulata,the prokaryotic and eukaryotic expression of spm-N gene was carried out in the experiment.With the specific primers,T.annulata spm-N gene was amplified by PCR,and was merged to prokaryotic expression vector pET30a(+)or eukaryotic expression vector pGBKT7.Then BL21(DE3)competent cells were used in prokaryotic expression plasmid transformation,The recombinant protein was expressed by IPTG-inducible promoters and collected as secreted form,which was to be used as antigen latter,and an indirect ELISA method was constructed to detect the antibodies of T.annulata,by optimizing reaction conditions of ELISA.This method did not show interreaction with the positive sera of piroplasma,such as T.sinensis,T.sergenti,Babesia bovis and so on,which indicated that the ELISA method had good specificity and immunogenicity.Besides,it also had good repeatability and sensitivity.So it provided a convenient and rapid serological diagnosis method for the rapid diagnosis and epidemiological investigation of the T.annulata.Eukaryotic expression plasmid was transformed to yeast strains Y187 competent cells,then it was detected that the self activation activity of spm-N protein in yeast strains,the effect on yeast growth and its expression in yeast.The results showed that the recombinant plasmid can be correctly expressed in yeast cells,and its expression did not affect the growth of yeast cells,and there was no toxicity to yeast cells and no activation activity in yeast cells.Visibly,PGBKT7-spm-N can be used to screen the materials interacted with the spm-N proteins from T.annulata.