Tandem Expression And Immunogenic Analysis Of Theileria Annulata Surface Antigen Proteins

Tropical theileriasis is an acutely, febrile tick-borne blood protozoa disease caused by Theileria annulata which parasitized on red blood cells, lymphocytes and macrophages of cattle. In addition to the direct cause of the diseased animals mortality, this disease reduces body weight and milk production, it lowers the resistance to other diseases, and leads to the onset of other illnesses and induces this disease.these circumstances not only seriously affected the development of animal husbandry, but also caused economic losses. Theileria annulata merozoite surface antigen, schizonts surface antigen and sporozoite antigen has been confirmed as having good immunogenicity. Given what are characterized by polymorphism and infection immunity in parasitic antigen gene. The single antigen biologicals of Theileria annulata produced incomplete protection in immunization reaction. So there is not high sensitivity and specificallity when detecting antigen. This study investigates further the three surface antigen of Tropical theileriasis. This aimed to explore the immunogenicity of the tandem surface antigen proteins. To lay a basis on the development of Tropical theileriasis subunit vaccine.In this study, bioinformatics software was used to analyze the recombinant Tams1-Spag1, Tasp-Spag1 gene, predict the main characters and good immunogenicity. Based on this, the recombinant plasmid pET-28a-Tams1-Spag1, pET-28a-Tasp-Spag1 was successfully constructed, and then this two recombinant plasmids was transformed into E.coil BL21(DE3). After the inducible expression by IPTG. the reactionogenicity of tantem protein is detected by Western blot technique. we have purified the recombinant protein, and then used recombinant protein as antigen to immune mice. Take the serum from mice to estimate the immunogenicity. We want to identify the immunogenicity of recombinant proteins. Based on the bioinformatics analysis, the recombinant protein tams1-spag1 contains 219 amino acids and belongs to stab hydrophilic non-secreted protein. It contains 13 Glycosylation sites and 11 Phosphoric sites, 2 low complexity domain and NOT homologous regions. There may be 6 B- cell major epitopes domain and 10T-cell major epitopes domain and two cross-reactive epitopes. The recombinant Tasp-spag1 contains 236amino acids and belongs to unstab hydrophilic nonsecreted non-transmembrane protein. It contains 33 Glycosylation sites and 21 Phosphoric sites, 3 low complexity domain and Sorb, NL homologous regions.There may be 10 B-cell major epitopes domain and 7 T-cell major epitopes domain and two cross-reactive epitopes. The predictab results indicate that two different recombinant proteins have good immunogenicity in theory. The result of SDS-PAGE and Western blot showed that the target protein was obtained with a molecular weight same as the expected size, The conditions of induced expression of recombinant plasmid were optimized. We find that this proteins were insoluble and existed in the form of inclusion body. The experimental result indicated that the maximal quantity of expression of recombinant Tams1-Spag1 protein was confirmed when the concentration of IPTG was 1.0 mmol/L and OD600=1, the induced time was stay overnight. The maximal quantity of expression of recombinant Tasp-Spag1 protein was confirmed when the concentration of IPTG was 0.9 mmol/L and OD600=1.2, the induced time was stay overnight. First, the target protein were purified by cutting the gel slices that contained the right bands which were stained by KCl solution. second, the recycle protein were recovered by electrical elution. Western blot assay indicated that this two recombinant protein were not denaturated with good reactionogenicity. Mices were immuned by the purified protein emulsified with Freund’s adjuvant. The serum of mice is detected by ELISA and Western blot. The results showed that this two recombinant protein can all stimulate specific antibody production after the immunity. This serum could specifically react with the vaccine protein of Tropical theileriasis. The study indicated that this two tandem protein both have better reactionogenicity and some immunogenicity.