Studies On The Purification And MRNA Differential Display Of Drug Resistance Of Eimeria Tenella

Single oocyst isolation and single sporocyst cloning of Eimeria tenella. The oocysts in faeces of the chickens were identified under microscope after saturated sodium chloride solution floating. Oocyst of E. tenella was isolated and purified by the single oocyst isolation technique, and the success rate was 38%. Oocyst of E. tenella was cloned by the single sporocystcloning technique, and the success rate was 28.6%. The homogenes strains of four diferential strains were respectively got.Purification of merozoites of Eimeria tenella. Merozoites from four strains of E. tenella were purified by serial methods, including the standard sample sift filteration, centrifugal washing, differential velocity centrifugation, and DEAE-52 cellulose column chromatography. 1.85 X 107 merozoites were obtained from 30 chickens, 6.17 X 105 merozoites from each chicken.Differential Display PCR of mRNA from merozoites of sensitive strain and resistant strain. In order to understand the molecular mechanism of drug resistance and to set up a molecular test of drug resistance in the laboratory, the differential expression between merozoites mRNA of maduramisin sensitive and maduramisin resistant strains of E. tenella was detected with the technique of random mRNA Differential Display. The correlationship between differential expression patterns were studied, the differential expression DNA fragments were identified by reverse Northern dot hybridization, then the possible functions and the relationship of the differential DNA fragments to drug resistance were predicted. Four differential expression fragments were cloned with PMD-18T vector and sequenced. One DNA fragment, which was similar to microneme-5 gene, was found. One DNA fragment, which was similar to surface antigen-17, 16 genes, was found. The other 2 differential expression sequences were unknown, whose homologous genes were not found in GenBank by Blast analysis. The biological traits were related to the genes from mutation and transformation, transcription, modification after transcription, alternative splicing, transport to cytoplasm, translation, modification after translation and so on. Results showed that the drug resistant trait was the ultimately result of a series of gene expression process and factors, affecting this serial process. This indicated that drug resistance was a complex biological process.Identification of the drug resistant strain by the molecular biological technique. The sense and anti-sense primers were designed according the special sequence from the resistant strain by the result of the differential display PCR. The total RNA was purified with Trizol from merozoite of 1 mduramisin resistant E. tenella strain and 3 differntial maduramisin sensitive E. tenella strains. The DNA was purified with DNA purification kit from oocysts of the same 4 differential strains. The drug resistance could be identified by RT-PCR. It was unanimous to the test results in chickens. But it could not be identified by PCR with DNA template.