The Prokaryotic Expression Of Eimeria Acervulina 3-1E Gene And Preparation Of Its Polyclonal Antibody

Chicken coccidiosis is one of the most severe diseases to chicken industry, The problem of death, productivity impairment, due to the outbreak of coccidiosis, brought huge economic loss to poultry industry. Nowerdays, the prophylactic measures against chicken coccidiosis is mainly depond upon drug therapy. However, for the enormous research and development expenditure of new medicine due to drug resistance of coccidian, treatment fee for utilization of a great amount of medicine in practice, and medicinal residue in chicken, these restrained the application of drug therapy. At present, the development of coccidiosis vaccine with modern genetic engineering techniques became the hot point at the field of coccidiosis control, thus it got more and more important to find and identify the antigen gene of coccidian.Specific primers for 3-1E cDNA of Eimeria acervulina were designed and synthesized according to the previously reported nucleic acid sequence of 3-1E cDNA of E. acervulina. The 3-1E cDNA of Shanghai Strains of E. acervulina was amplified by RT-PCR method and sequenced. The result showed that it was 513bp in longth, which contained an open reading frame. Comparing with 3-1E gene of other reference strains of E. acervulina US strain, E. tenella GS strain and E. acervulina QH strain it had the homology of 100%,99.6% and 99.2% in nucleotide sequence, and 100%,99.4% (site of 98 changed from valine to isoleucine), 98.3% (site of 95 changed from eonine to isoleucine, site of 96 changed from arginine to glycine, site of 98 changed from valine to isoleucine) homology in amino acid sequence. The prokaryotic expression plasmid pet30a/3-1E was constructed and transformed into BL21 cells. The fusion protein was expressed at 37℃induced with IPTG. SDS-PAGE and western-blot analysis showed that the fusion protein had a molecular weight 26 Ku, which counted 35% of total bacterial protein. The fusion protein was purified by QIAGEN Ni-NTA resin and used to immunize rabbit for preparation of polyclonal antibody. Agar-gel diffuse experiment and Western-blot showed that polyconal antibody had good reactivity with the 3-1E fusion protein.This experiment successfully cloned the 3-1E gene of E. acervulina, carried out the prokaryotic expression and purification of the fusion protein, and prepared the rabbit anti-E. acervulina 3-1E polyclonal antibodies. it offered solid foundation for the research of biological characteristics and applications of 3-1E gene.