微小隐孢子虫端粒序列及端粒酶活性的研究

为了探讨我国微小隐孢子虫(C.parvum)的端粒序列,试验根据国外已报道的端粒重复序列5’-CCTAAA-3’设计寡聚核苷酸探针(CCTAAA)5,并用地高辛标记,C.parvum基因组DNA经Bal31-HindⅢ酶切后进行Southern印迹杂交鉴定;并在此基础上,采用改进的端粒重复序列扩增(TRAP)法首次对子孢子和卵囊时期C.parvum的端粒酶活性进行检测。结果表明:C.parvum基因组DNA与探针(CCTAAA)5杂交,获得了较好的杂交条带,随着Bal31-HindⅢ酶切时间的延长,杂交信号逐渐减弱,说明C.parvum端粒序列定位在染色体的末端,与国外报道的C.parvum端粒序列一致,为5’-CCTAAA-3’;检测C.parvum端粒酶活性时发现,子孢子时期端粒酶具有活性,卵囊中未检测到端粒酶活性。

Research on the telomeric sequences and telomerase activity of Cryptosporidium parvum

To investigate the telomeric sequences of Cryptosporidium parvum(C.parvum) in China,the oligonucleotide probe(CCTAAA)5 marking with digoxin was designed according to the telomeric repeat sequences 5′-CCTAAA-3′ reported in foreign countries.The genomic DNA of C.parvum was digested with Bal31-HindⅢ,and was identified by hybridization of Southern blot.On this basis,the telomerase activities of C.parvum in the sporozoite and oocyst stages were detected using the modified TRAP.The results showed that the genomic DNA of C.parvum could hybridize with oligonucleotide probe(CCTAAA)5,and the hybridization band was found clearly.The hybridization signal gradually weakened with the extension of Bal31 digestion time.The result indicated the telomeric sequence located at the end of the chromosome was identified as 5′-CCTAAA-3′,which was in accordance with the telomeric sequences of C.parvum reported in foreign countries.It was confirmed that the telomerase activity was found in the sporozoite stage,which was not found in the oocyst stage.