Amplification And Sequence Analysis Of Single-Chain Antibody V_H And V_L Genes Of Eimeria Tenella Sporozoite

Coccidiosis in Chicken is a parasitic protozoa disease which was caused one of seven coccidium infection in different part of intestinal tract cell. Now, preventive treatment of chicken coccidiosis is mainly depended on anti-coccidia drug, but the long term using made the coccidium emerge drug tolerance, meanwhile, the drug residue of animal product effect the health of people, so, the use of immune preventive treatment to replace the drug treatment become the inevitable coccidiosis control, In this study, Eimeria tenella (Nong An strain)monoclonal antibody was prepareed and the specificity of E.tenella VH and VL genes were cloned and sequence analysis, lay the foundation of the specificity of E.tenella single-strand antibody gene and restructuring toxins construction,expression,activity determination between the specificity of E.tenella single-strand antibody gene and pseudomonas aeruginosa beta-exotoxin PE40, The restructuring toxin is expected to become the new tools against Eimeria tenella disease.1.The preparation of Specificity single antibody of Eimeria tenella sporozoite.To prepare the soluble antigen of Eimeria tenella by egg capsule re-strong% collect,vitro spore,purification,grinding,excystation,purification by DEAE-52,repeat freeze thawing, Ultrasound crackincentrifugation. The density of Antigen samples is 818.4pg/mL which was Measured by ultraviolet method. To established the monoclonal antibody lines the soluble spores antigen, use this to detect supernate of hybridoma cell, to gain the positive hybridoma cell, then cloned and expand cultivation.2. monoclonal antibody light and heavy chain variable area gene cloning and sequence analysis of Chicken Eimeria tenella sporozoiteRecovery hybridoma cell which can secrete monoclonal antibody of Eimeria tenella, detect the activity of the hybridoma cell with Agarose diffusion method when the cell Reach best growth state. Using the Trizol method to extract total RNA from hybridoma cell, the result showed that the total RNA of hybridoma cell was extracted succeed, A26o/280=1.96; The Specific Primers were designed and synthesized according to the published single-chain antibody VH and VL sequence of Eimeria acervulina in GenBank (accession numberAJ298107), using the extracted RNA as templates, with RT-PCR one-step method to amplificate purpose gene,connected target gene and pMD-18T vector, after PCR identification, to sequence the positive recombinant plasmid, the result showed that the gene of Light chain variable area are 321 bp, the heavy chain variable area are 369 bp, Sequencing results showed that accord with chicken antibody light chain variable area and heavy chain variable area genetic characteristic. But the CDR gene which was combined with epitope was changed, there was gene deletion phenomenon in Light chain variable area genes.