Cloning Of The Telomeric DNA And TERT Gene And Detection Of Telomerase Activation In Trichinella Spiralis

Trichinella spiralis is one of the most wide spread parasites and can infect humans and more than 150 species of mammals all over the world. Infection of animals and humans by Trichinella spiralis remains a serous public threat in both developed and developing countries. 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. T.spiralis as the research target in this study,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 T.spiralis telomeric DNA was identified. At the same time, pairs of degenerate primer were designed on the basis of other nematode TERT sequence and T.spiralis TERT full-length cDNA cloned successfully and the composition of T.spiralis TERT gene was analyed with protein and nucleic acid sequence analysis software also. At last, the telomerase activity in ML,Ad and NBL phase was detected using the designed primers according to the infomation of T.spiralis telomeric DNA. Data presented in present study provide a biochemical and structural basis for future studies on T.spiralis telomerase and will potentially lead to a better understanding of the mechanism of growth and aging in other nematode . The detailed research contents and results as follows:Identification of telomeric repetitive DNA sequences of T.spiralisThe genomic DNA was extracted from TIANamp Genomic DNA Kit and was digested with PstⅠand 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'-TTAGGC-3'was the T.spiralis telomeric DNA. In order to indentify the results, oligonucleotide probes (5'-TTAGGC-3')5 which was labeled with digoxin were hybridizated with T.spiralis genomic DNA and the T.spiralis genomic DNA which was digested with BAL31 and EcoRⅠin different time. The results of it indicated that the genomic DNA of T.spiralis 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 T.spiralis was 5'-TTAGGC-3'.Cloning and analysis of T.spiralis TERT sequenceDegenerate primers were designed based on the sequences of the C.elegans,C .remanei, C.briggsae 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 T.spiralis TERT sequence,initially two pairs of degenerate PCR primers were used to amplify a fragment of the T.spiralis 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 T.spiralis TERT open reading frame encoding a protein of 1201 amino acids with a predicted molecular mass of 139 kDa. Pairwise alignment of full length T.spiralis TERT with other previously characterized TERTs using the DNAMAN software revealed high sequence identitities with C. elegans. 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 T.spiralis TERT. Like other TERTs, T.spiralis 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.Detection of telomerase activity T.spiralis in different development stagesA pairs of primers were desiged according to T.spiralis telomeric DNA sequence. The telomerase activity of T.spiralis 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 Ad and NBL,while not detected in ML phase.Inhibition of matrineon telomerase activity in different developmental stages of T.spiralisTelomerase activity of T.spiralis were detected by different concentrations of matrine using TRAP assay . The results showed that the concentration of matrine in 0.5mg/mL and 1.0mg/mL, the different stages of the parasite T.spiralis had been no inhibitted in telomerase activity, and matrine at a concentration of 2.0mg/mL, 4.0mg/mL When muscle larvae of T.spiralis no inhibitory effect of telomerase activity, and significantly inhibited T.spiralis adult, newborn larvae of T.spiralis slight inhibition.