Clone And The Research Of The Immunity Of E.tenella 5401 Gene

Coccidiosis is one of the most important parasitic disease in chickens. The disease has high incidence and high mortality,resulting in a great deal of economic losses in the poultry industry. Chemotherapy is the major means for the control of E.tenella infection. With the development of the resistant strains as well as the formation of green food concept,a better control method is expected. Research on vaccine against coccidiosis started at 1960s, 4 life vaccines have been utilized in chicken industry with some shortcomings. Subunit vaccines produced by gene technology was found to have partial protective effect at the end of 1980s. In order to provide a basis for development of a better gene engineered vaccine, this research focused on the 5401 gene.Oocysts of E.tenella were propagated, isolated and sporulated using standard procedures, the sporulated oocysts were pelleted by centrifugation, surface-sterilised using sodium hypochlorite, washed several times in water and stored in 2.5% potassium dichromateat 4'C.RNA isolated from sporulated E.tenella oocysts was used as a template for cDNA synthesis by RT-PCR. The gene-specific primers was synthesized containing the upstream and downstream of 5401 gene sequence, which also involved an oR I and Sal I cleavage site respectively at its upper or lower primer in order to be easily ligated to the plasmid later. The upper primer sequencers' ) TCG AAT TCA TGG GCC AAA CCG GAG AGO AG ; The lower primer sequence: (5') GCC GTC GAC CTA CTG GAT GTC ACC ACT GTC TG. The PCR product of around 881bp was purified and ligated into T-vector(Promega) and transformed into E.coli DH5- a competent cell. Identified by PCR and cleaved with endonucleases, the positive clone was used to determine its nucleotide sequence. Results indicated that, the full-length of 5401 gene was 864 bp in E.tenella ZJ strain, it's the whole ORE Compared with the 5401 sequence reported by Danforth (1989), mutations occurred at 2 bases of 2 regions and their homology was 99.8%. The homology of the amino acid was 99.3%.The cDNA inserts of positive cDNA clones was subcloned into suitable expression vectors (PET-30a) by standard procedures of restriction endonuclease digestion, DNA fragment isolation and ligation. Direct expression was achieved by insertion of a synthetic linker carrying an ATG codon.In the expression experiment, the 5401 antigen was expressed by the induction of IPTG in the form of a dimer protein. Cultures of E.coli BL21(DE3) carrying recombinant plasmids PET-30a-5401 were grown in LB supplemented with 100 n g ml"1 Kan. Cultures were shaken at 37癈 for 2hs to density of about 5X 108 cells ml'1 . IPTG was added to 0.5mM, and cultures were shaken at 37? for 2-6hs. Cells were harvested by centrifugation and solubilized in SDS-PAGE sample buffer and heated for 5 min at 100癈 and analyzed by SDS-PAGE. The result of SDS-PAGE shows that the fusion protein was about 66.2 kDa, it's a dimer protein. The product of the fusion protein reachs a high level 6hs after IPTG added. The specific protein band was detected by Western-blot.The specific protein band was detected by Western-blot.In the animal immunity experiment, 3-day-old chicks were randomly divided into six groups with 40 birds in each group. Group A chickens were kept as unimmunized controls. Group B were vaccinated subcutaneously (s/c) on the breast region at 3 days of age with 100u.g recombinant 5401 antigen and a booster dose of 100u.g same antigen was given at 17 days of age; Group C were vaccinated subcutaneously at 3 days of age with lOOug recombinant 5401 antigen and ginsenosides (0.25mg each chick) and a booster dose of same same antigen and ginsenosides were given at 17 days of age; Group D were vaccinated s/c with lOOug recombinant 5401 antigen and ginsenosides (O.SOmg each chick ) and a booster dose of same same antigen and ginsenosides were given at 17 days of age; Group E were vaccinated s/c with lOOug recombinant 5401 antigen and ginsenosides (0.50mg each chick) and a booster dose of same antigen and ginsenosides were given...