| It is important that telomerase expression during proliferation of eukaryotic cell growth, and it has been identified that it could impair the proliferation of tumour cell and disintegration of protozoan by suppression of telomerase activity with the specific inhibitors of telomerase. Therefore, studying the activity of telomerase will provide a new method to treatment parasitic disease. The telomerase activity of many parasitic protozoa has been detected by now. However, the activity of Cryptosporidium parvum (C.parvum) has not been reported. In present study, the telomerase activity of C.parvum sporozoite and oocysts were detected by modified telomere repeat amplification protocol (TRAP), as well as the inhibition action that sophocarpidine made on the the telomerase activity. At the same time, the telomeric repetitive DNA Sequences of C.parvum was identified.Identification of telomeric repetitive DNA Sequences of C.parvum According to the result of Alan T. Bankier reported that 5'- CCTAAA-3'was the telomeric repetitive DNA Sequences of C.parvum, the oligonucleotide probe was designed. Then the genomic DNA of C.parvum was hybridizated with oligonucleotide probe. The result indicated that the genomic DNA of C.parvum could hybridizate with oligonucleotide probe and zone of hybridization could be seen clearly. With the time of Bal 31 digestion delay, the zone of hybridization on the nylon membrane disappeared. The result showed that exonuclease Bal 31 hypersensitive to the telomeric repetitive DNA Sequences of C.parvum, this verified that the telomeric repetitive DNA Sequences of C.parvum was 5'- CCTAAA-3'. The identification of telomeric repetitive DNA Sequences was the foundation for the detection of telomerase activity.Detection of C.parvum telomerase activity According to the modified TRAP, forward primer p1 and six reverse primer p2 were designed. Among the primers, the forward primer p1 was consisted of a telomere repetitive sequences of C.parvum at the 3′end and 9bp random sequences at the 5'end. The reverse primer p2 consist of an anchoring sequence at the 5′end and two telomeric complementary repetitive sequences with base sequences permuted at the 3′end. Compared to other primers, the result indicated that the C.parvum telomerase activity was highest when amplificated by reverse primer p2-6. It indicated that this primer could be more efficiently combined to the extend products of telomerase. At the same time, two negative controls were established during TRAP. The first negative control was cell extracts pretreatmented by 50μg /mL of RNase A for 30 min at 37°C, the second one is the extracts replaced by CHAPS solution, it ensured that the TRAP products could represent the activity of telomerase. The result showed that high telomerase activity was detected in C.parvum sporozoite and there was no telomerase activity been detected in C.parvum oocysts. This indicated that telomerase could repair the damage of terminal end of chromosome so that maintain stabilization and integrity of C.parvum chromatosome.In summary, these results identified that telomeric repetitive DNA Sequence of cryporidium parvum was 5'- CCTAAA -3'and confirmed the telomerase activity was presented in sporozoite not in oocysts. Moreover, telomerase was slightly inhibited, and telomerase activity decreased unobviously in sophocarpidine inhibition test.In sophocarpidine inhibition test, inhibition action that sophocarpidine with different density made on C.parvum telomerase activity was detected by TRAP. Telomerase was acted by Sophocarpidine whose density was 0.5 mg/mL,1.0mg/mL,2.0mg/mL. Of the total, telomerase activity was slightly inhibited by Sophocarpidine of 1.0mg/mL,2.0mg/mL but not the others ,but the telomerase activity decreased unobviouslyThis study showed that telomerase provided a potential target to prevent and treat C.parvum disease. Cryptosporidiosis and other protozoon disease can be prevented by adjusting the activity of telomerase.
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