Cloning And Detection Of Drug-resistance Gene M₂₀ Of Eimeria Tenella

Coccidiosis caused by protozoan parasites of parasitic intestinal epithelial cells in the chicken is harmful to 15 to 50 day-old birds. It is a widespread disease of high mortality rate. At present, drug prevention is currently still the main treatment. Because long-term drug misuse is not standardized, resulting in general drug-resistant strains. All drugs-resistant strains were isolated from the farm used in treatment. Occurrence interval of drug-resistance, the levels was reduced and increased. The resistance was changed from single drug-resistance to multiple drug-resistance. Drug-resistance has become the most serious problem of the prevention and treatment of coccidiosis, but the mechanism is unclear. The detection of a particular drug-resistance gene can monitor the spread of drug-resistance and guide strategy of drugs.Suppression subtractive hybridization (SSH) of Eimeria tenella between drugs-resistant strains and their parents strains. Full length sequences of some drug-resistance genes were obtained by RACE method. A new gene of M20 among them was cloned and expressed by prokaryotic system. A new method of detecting drug-resistance of Eimeria tenella was established via Real-time fluorescence quantitative PCR, and the result of real time PCR were verified by animal test.1. Cloning and expression drug-resistance gene M20A number of ESTs sequences were obtained by suppression subtractive hybridization (SSH) of Eimeria tenella between drugs-resistant strains and their parents strains.According to one of ESTs,total RNA of the sensitive strain was isolated,and then the full-length cDNA sequence of gene was cloned by RACE.The length of gene was 847 bp,containing a 525 bp open reading frame (ORF),encoding a deduced protein of 174 amino acids protein of 19 KDa.It is revealed that the gene transcript level in the unsporulated oocysts sensitive strain was much higher than other developmental stages (sporulated oocysts,sporozoites and merozoites).M20 was ligated with pET28b prokaryotic vector,and constructed recombinant plasmid pET28b-M20. Western blot analysis showed that the protein could be specifically recognized by polyclonal antibodies against E.tenella.2. Establishment of Real-time PCR to detect drug-resistance of EimeriaAccording to M20 gene specific primers of full length sequence, SYBR Green and TaqMan Probe Real-Time PCR were established for a simple, rapid detection of drug-resistance of coccidian. The results showed that both methods have good specificity and repeatability. The M20 levels of the anti-diclazuril strain,the anti-Maduramycin strain and the drug-sensitive strain were detected by SYBR Green which is inexpensive. The result showed that M20 level of the sensitive strain was significantly higher than two resistant strains, indicated that the establishment of Real-time PCR for detection is feasible.3. Application real time PCR on detection drug-resistance of the Eimeria field strainM20 level of the field strain obtained by single oocyst isolation technique was detected via SYBR Green Real-Time PCR. At the same time, the anti-diclazuril strain, the anti-Madura strain and the drug-sensitive strain were regarded as a reference. The result showed that M20 level of the field strain were higher than two other strains, indicated that the isolated field strain was drug- sensitivity. In order to verify the result of Real-time PCR, the animal test was carried out. The result showed that the isolated field strain was sensitive to maduramycin, decoquinate, diclazuril and salinomycinthe. The result of animal test was consistent with the Real-time PCR.In summary, a new drug-resistance gene M20 of Eimeria tenella was cloned and expressed. The anti-diclazuril strain, the anti-Madura strain and the drug-sensitive strain were regarded as a reference, and SYBR Green Real-Time PCR for drug-resistance detection was established by target gene M20. Real-time PCR was verified the accuracy by animal test. Real-time PCR detection method has overcame some shortcomings, such as a long period of research time, high costs. The study provides a new way of thinking for the future detection of drug-resistance.