Cloning,prokaryotic Expression And Immunoreactivity Of The Truncated M2AP Gene Of Toxoplasma Gondii

Toxoplasma gondii is an obligate intracellular parasitic protozoan,which can infect humans and almost all warm-blooded animals.It poses a great harm to human health and animal production.It is of great significance to develop new diagnostic methods for toxoplasmosis to ensure public health safety and sustainable development of animal production.T.gondii microneme protein 2 associated protein(M2AP)is secreted by the microneme organelles.It can participate in the sliding movement and invasion of the parasite,and play an important role in the process of parasite invasion of host cells.Previous studies have shown that it can react with chicken and rabbit anti-Toxoplasma sera and is a potential diagnostic antigen.However,whether M2AP reacts with the sera of other animals infected with T.gondii remains unknown.In addition,T.gondii M2AP has high sequence similarity with that of other Eucoccidiida protozoans,which suggests that it is likely to cross-react with the animal sera infected with other Eucoccidiida protozoans.Bioinformatics shows that T.gondii M2AP has a species-specific region.In this study,the reactivity and diagnostic specificity of this species-specific region were studied.Based on the T.gondii M2AP gene sequence published in the NCBI database,this study analyzed the physical and chemical properties,signal peptides,hydrophilicity/hydrophobicity,surface accessibility,antigen index,antigen epitopes and homology of the truncated M2AP protein of T.gondii by using different bioinformatics methods.Then,prokaryotic expression and purification of the specific region of T.gondii M2AP were performed,and its reactions with Toxoplasma antibody/positive sera of several animals and positive sera of other Eucoccidiida protozoans were analyzed by Western Blot method.The results showed that the amplified species-specific sequence of T.gondii M2AP was identical with that available in database.The theoretical molecular mass of the corresponding truncated M2AP protein is approximately 9.7 k Da,the total average hydropathic index is 0.897;the analysis showed that the protein contains a complete antigen epitope composed of 26 amino acids,and the antigenic index is relatively high.Therefore,it was predicted that the truncated M2AP protein has good reactogenicity.The truncated M2AP gene was successfully amplified,cloned and expressed prokaryotically.Western Blot results showed that the recombinant truncated M2AP protein can react with goat and rabbit anti-Toxoplasma antibodies,and Toxoplasma positive sera of pigs and alpacas,but not with animal sera infected with Neospora and Eimeria tenella,indicating that it has good reactogenicity and specificity.In summary,this study cloned and expressed the species-specific sequence of T.gondii M2AP,and examined its reactogenicity and specificity.The results showed that the recombinant truncated M2AP protein had good reactogenicity and specificity,thus extended the host range of its applicability,which also provided foundation for developing new diagnostic methods for toxoplasmosis.