Cloning Of Surface Antigen Sag13 And Sag14 Gene From Eimeria Tenella And Expressing Them In Prokaryotic Expression System

The Eimeria species, causative agents of the disease coccidiosis, are seriously harmful to the poutry production. Drugs have been used to help control these diseases for a long time. However, there are issues with increasing development of resistance to many of the anti-coccidial drugs used to help control avian eimeriosis and public concerns about the use of drugs in food animals. Existing vaccines consist of live virulent or attenuated Eimeria strains. These live vaccines can have problems with safety, short shelf-life and large-scale production; therefore there is interested in devising new vaccines using defined recombinant vaccines and/or DNA vaccines. Many recombinant antigens or DNA had been proved to induce protective immune responses, including some surface antigens and secrectory antigens have been characterized from organelles such as micronemes, rhoptries and refractile bodies. The antigens SAG13 and SAG14 which expressed in both the sporozoite and second merozoite developmental stages are glycosylphosphatidylinositol (GPI)-linked surface proteins in the coccidian parasite Eimeria tenella. The biological role(s) of these proteins and the effect(s) of variant surface antigens are unknown. We choose the surface proteins (SAG13 and SAG14) to research in E. coli and hope the study can provide some evidence for coccidiosis.(1) Cloning and sequencing of sag13 and sag14 genesOne cDNA fragment of sag13 and sag14 genes were amplified by RT-PCR with primers which were designed to remove signal sequences according to GenBank. Recombinant plasmids pGEM-sag13 and pMD19-sag14 were constructed. The ligation products were transformed to competent cells of E.coli host strain Top10. Clones were cultured and the recombinant plasmids were prepared as template for automatic sequencing. The nucleotide sequences were analysed by DNAman sequence software. The results indicated that the gene fragment encoding sag13 was 732bp in length. Compared with database in GenBank, sag13 shared 100% on DNA sequence homology. The gene fragment encoding sag14 was 738bp in length. Compared with database in GenBank, sag14 shared 100% on DNA sequence homology. We also analyse the primary structure and secondary structure of SAG13 and SAG14 by SOPMA software.(2) Constructing and expressing of prokaryotic expression vector pET30-sag13 and pET30-sag14The recombinant plasmids pET30-sag13 and pET30-sag14 were constructed by cloning sag13,sag14 genes into prokaryotic expression vector pET-30a(+) and expressed in E.coli host cells BL21-Gold (DE3). Valued SDS-PAGE by Bandscan analytic software, the molecular weight of recombinant proteins were about 33.5 kD and 36 kD which were close to theoretical ones (31.2kD,31.4kD). Then they were identified by westem blot after purified using the nickel column chromatography. The expression levels were optimized by manipulating the host strain and the time of harvest. In normal LB media, the recombinant strain SAG13 was incubated by 0.4mmol/L IPTG for 2h in 30℃before harvesting cells. The yield of soluble recombinant SAG13 was up to 50.5% of the total soluble bacteriaI protein in the cell lysate. The recombinant strain SAG14 was incubated by 1.0mmol/L IPTG for 4h in 30℃before harvesting cells. The yield of soluble recombinant SAG14 was up to 66.7% of the total soluble bacteriaI protein in the cell lysate.