Development Of Diagnostic Method And DNA Vaccine For Bovine Theileriosis Sergenti

Bovine theileriosis sergenti, caused by Theileria family, Theileria genus, Theileria sergenti, is one of the blood protozoal disease, characterized by hyperpyrexia, anaemia, bleeds, jaundice, becomes thin and the enlargement of lymph nodes of body surface, especially jeopardize foreign ox, pedigree cattle and improving bastard ox seriously.Theileriosis sergenti was firstly found in ox body of the Far East Area of Russia byЯкимовandДегтерев(1930), and identified as a new species, then appeared in countries such as Japan, South Korea, Thailand, Holland, Italy, China. In recent years, it occurred frequently because of the rapid development of ox industry and trade activity, the epidemic area of this disease is expanded gradually, and has already been becoming one of the serious diseases threatening development of ox industry. But the culture in vitro of the causative agent has not succeeded yet, which hindered the further investigation of this disease, there has no effective vaccine to control the disease so far. Therefore, utilize genetic engineering is the only feasible way to prepare antigen proteins for diagnosis and vaccination. Surface proteins of Theileria sergenti include P23, P32, P33, P34, P35 ,P45, etc. P33 is one of the major protein on the surface that can induce strong immune response in the stage of rhodocyte infected with Theileria sergenti and it is one of the main vaccine candidate antigen. DNA vaccines is the eukaryotic expression plasmid that encode protective antigen gene of the pathogenic microorganism, after vaccinating animals by skin or intramuscular injection, it expresses proteins in the cells, which combine with MHC-Ⅰand be presented to the cells' surfaces, stimulate organism to produce immune response . The expression of the interest protein in the cells of immunized animals can form natural structure and retain the maximal immunogenicity to induce high-level humoral immunity and cellullar immunity. So the following investigation were carried out: Cloning and sequence analysis of P33 gene of Theileria sergenti. we amplified P33 gene from domestic Theileria sergenti, then the gene sequence and heredity evolution analysis indicated that the length of P33 gene was 868bp, and encoded 283 amino acids, there were two potential glycosylation sites, which situated in 42-44aa and 106-108aa; the nucleotide sequence homology was 99.4%,88.0%,88.1%, respectively, with Korea, Japan, Russia strain, and amino acid sequence homology was 98.9%,86.2%,86.6% respectively. The analysis of heredity and evolution indicated that the strain was originated from Korean strain.The prokaryotic expression of P33 gene of Theileria sergenti. P33 gene was subcloned into pET-28a expression vector to construct recombinant plasmid pET-28a-P33, which was confirmed by PCR, sequencing and restriction enzymes analysis. The analysis of SDS-PAGE indicated that the gene has been expressed in E. coli. The recombinant protein existed as inclusion bodies with molecular weight of 30.3 kD. Western blotting analysis indicated that the expression protein can reactive with specific antibody.Establishment of PCR diagnostic method. According to the conserved sequence of P33 gene of Theileria. sergenti Chinese strain, the primer was designed, and the PCR diagnostic method for Theileriosis sergenti was established. A gene fragment with the length of 499 bp can be amplified by this method and the homology was 100%. The positive rate was 72% (54/75) in 75 samples by the established PCR method , whose sensitivity could reach about 18 fg, and had no cross reaction with T. annulata,B. ovata,Anaplasma. It demonstrated that the PCR diagnostic method has highly specificity and sensitivity, and could be used in diagnosis and epidemiologic survey for Theileriosis sergenti .Establishment of the ELISA diagnostic method for Theileriosis sergenti. P33 recombinant protein of Theileria. Sergenti was used as antigen to develop a new ELISA diagnostic method. The results of cross reaction showed that this method had no cross reaction with positive serum of T.annulata, B.ovata, Anaplasma, Eperythrozoon and Neospora caninum. 60 serum sample has been detected and the positive rate was 70.0% by ELISA with coincidence rate of 96.7%, compared with the positive 66.7% by LAT and 43.3% by blood smear (BS).The research of DNA vaccine on Theileria. Sergenti. Two pairs of primer were designed and synthetized according to P33 and P23 gene sequence of Theileria. Sergenti in Genebank, pMD18-T-P33 and pMD18-T-P23 were constructed, respectively. The two fragments were subcloned into pVAX1 to construct recombinant plasmid pVAX1-P33-P23, which was transfected into Hela cells and the expresssed recombinant fusion protein was demonstrated by Western blotting analysis. Animal experiment indicated that recombinant plasmid could induce response of cellular and humoral immunity in mice. The mice was inoculated to indicated that group was vaccinated with pVAX1-P33-P23 elicited CD4+ and CD8+T subtype cells higher than the groups was vaccinated with pVAX1-P33and pVAX1-P23, significantly. Each group , specific antibodies were elicited first week after vaccinated ,and the antibodies higher than the control group(P<0.01), and increased gradually. The antibodies levels of pVAX1-P33-P23 was higher than that of pVAX1-P33and pVAX1-P23 , the second week after vaccinated and remain different during experiment, significantly.In a word, a sensitive, specific and convenient PCR and indirect ELISA methods were established for Theileriosis sergenti diagnosis. The eukaryotic expression plasmid pVAX1-P33-P23 of Theileria. Sergenti was constructed, which could induce response of cellular and humoral immunity in mice. This study has provided a foundation for prevention and control of bovine Theileriosis sergenti.