Cloning Of The Telomeric DNA And TERT Gene And Detection Of Telomerase Activation In Eimeria Tenella

Chicken coccidiosis is an important avian protozoan disease which seriously harm to the poutry industry worldwide. It was caused by various Eimeria spp. parasitized in intestinal mucosal epithelia, which are characterized with enteric pathological lesions. Among various Eimeria spp., E.tenella, which causes caecal coccidiosis, is highly pathogenic. Presently, the control of coccidiosis chiefly depends upon prophylactic chemotherapy with anticoccidial drugs. However, the emergence of drug resistance in coccidia is a great problem with most of the drugs, which, in due course, limits their use. Furthermore, drug or antibiotic-residue in the poultry product is potentially harmful to consumers. These limitations have necessitated the search for alternative Eimeria control measures. Telomere is a special nucleoprotein complex located at the ends of chromosomes that protects the termini from fusion, degradation, and recombination. Telomerase is a ribonucleoprotein complex responsible for extending the G-rich strand of telomere repeats composed of RNA and protein subunit. Telomere binding protein can adjust the length by adjusting the activity of telomerase. It has been becoming one of the spotlights of biological research to study on the telomeres and telomerase. The telomere and telomerase of parasites play a vital role in development, gene expression and variation, cell cycle regulation, gene recombination and life cycle composition and so on. E.tenella as the research target in this study, a powerful infectivity strain was isolated from Jilin province by single-oocyst isolation technique and its correlated bionomics was studied. On the basis of it, a libarary including telomeric DNA was constructed successfully. Then the probe was desiged according to orther parasites telomeric DNA and the positive clones which have telomeric DNA were screened from the constructed libarary, thus the E.tenella telomeric DNA was identified. At the same time, pairs of degenerate primer were designed on the basis of other coccidian protozoon TERT sequence and E.tenella TERT full-length cDNA cloned successfully and the composition of E.tenella TERT gene was analyed with protein and nucleic acid sequence analysis software also. At last, the telomerase activity in unsporulated oocyst, sporozoite and merozoite phase was detected using the designed primers according to the infomation of E.tenella telomeric DNA. Data presented in present study provide a biochemical and structural basis for future studies on E.tenella telomerase and will potentially lead to a better understanding of the mechanism of growth and aging in coccidian and in other apicomplexans. The detailed research contents and results as follows:1. Construction of E.tenella single-oocyst isolation strainThe samples were collected from the serious clinic morbility case which was confirmed as E.tenella infection observation by light microscope. In order to get a single pure line strain to study the E.tenella telometic DNA and TERT sequence, a single-oocyst isolation strain was isolated by single-oocyst isolation technique. Oocyst is ellipsoid, size (21.3±3.2)×(18.9±2.1)μm with a smooth, brown wall, without a micropyle, sporocyst residuum and oocyst residuum,with a polar granule. The sporocysts are oblong ovoid with stieda body. The shortest sporulatic time is 19 h. The minimum prepatent period is 118 h. At the same time, the result of PCR identified that single-oocyst isolation strain was E.tenella. Then the pathogenicity of it was studied, the result of it indicated that the E.tenella has high pathogenic, including their relative weight gain rates can fall 55.2% and the lesion score can reach 3.52, the mortality to chickens can reach 50%, when chickens are inoculated with a dosage of 4.0×104 sporulated oocysts each bird.2. Identification of telomeric repetitive DNA sequences of E.tenallaThe genomic DNA was extracted from fresh purified E.tenalla oocysts with chloridate and was digested with PstⅠa nd SmaⅠ, the same to pUC18 vector. The digested pruducts were extracted by phenol-chloroform then recoveried ethanol precipitation, the recoveried products was ligated with T4 DNA ligase, the ununited terminal was filled-in with Klenow fragment DNA polymerase I and the products was ligated with 0.1 U T4 DNA ligase at 4℃overnight. The positive clone which has telomeric DNA was screened by Southern blot and restriction enzyme disgestion to sequence. The results of sequencing indicated that 5'-TTTAGGG-3'was the E.tenalla telomeric DNA. In order to indentify the results, oligonucleotide probes (5'-TTTAGGG-3')5 which was labeled with digoxin were hybridizated with E.tenalla genomic DNA and the E.tenalla genomic DNA which was digested with BAL31 and EcoRⅠin different time. The result of it indicated that the genomic DNA of E.tenalla could hybridizate with oligonucleotide probe and zone of hybridization could be seen clearly. With the time of BAL31 digestion delay, the zone of hybridization on the nylon membrane disappeared. The result showed that the telomeric repetitive DNA sequence of E.tenalla was 5'-TTTAGGG-3'.3. Cloning and analysis of E.tenella TERT sequenceDegenerate primers were designed based on the sequences of the C.parvum, B.bovis, T.gondii TERT genes. The conserved regions were determined using the BlockMaker program, the degenerate oligonucleotide primers were derived from these conserved sequence blocks with online software CODEHOP. In order to isolate the E.tenella TERT sequence, initially two pairs of degenerate PCR primers were used to amplify a interior fragment of the E.tenella TERT inner sequence. On the basis of the sequence, both 3′and 5′-RACE were performed to isolate upstream and downstream sequences. Sequence analysis of the compiled DNA sequence demonstrated that the full length E.tenella TERT open reading frame encoding a protein of 1497 amino acids with a predicted molecular mass of 172 kDa. Pairwise alignment of full length E.tenella TERT with other previously characterized TERTs using the DNAMAN software revealed high sequence identitities with C.parvum. With conservation of previously identified motifs in the N-terminal region, C-terminal region and the central catalytic domain, motifs GQ, CP and QFP were found within the N-terminal of E.tenella TERT. Like other TERTs, E.tenella TERT contains the previously identified telomerase specific T motif within the central region of the protein followed by RT motifs 1, 2, A, B, C, D and E in the carboxyl terminal half.4. Detection of telomerase activity E.tenella in different development stagesFourteen days-old chickens are inoculated with a dosage of 1×105 sporulated oocysts each bird, E.tenella unsporulated oocysts, sporozoites and merozoite were collected. A pairs of primers were desiged according to E.tenella telomeric DNA sequence. The telomerase activity of E.tenella in different development stages were detected by the modified TRAP essay, the telomerase activity of sample which was digested with RNase and CHAPS were detected as negative control. High telomerase activity was detected in sporozoites and merozoites, while not detected in unsporulated oocyst phase.