[特异性锤头状核酶对蓝氏贾第鞭毛虫丙酮酸激酶mRNA表达的抑制作用]
[Inhibition of pyruvate kinase mRNA expression in Giardia lamblia by specific hammerhead ribozyme].
作者信息
Feng Xian-min, Cao Li-jing, Wang Feng-yun, Zhang Xi-chen, Lu Si-qi
机构信息
Department of Pathogen Biology, Jilin Medical College, Jiln 132013, China.
出版信息
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2010 Aug;28(4):257-60.
OBJECTIVE
To inhibit the expression of pyruvate kinase (PK) mRNA in Giardia lamblia by specific hammerhead ribozyme.
METHODS
The constructed hammerhead-GCV vector (pGCV-PKH) which aims to PK mRNA was electroporated into G. lamnblia trophozoites (group A). Electroporated trophozoites (group B) and normal trophozoites (group C) served as control Trophozoites in each group were collected at 24, 48, 72 and 96 h post-electroporation, respectively. The concentrations of trophozoites were calculated and the growth curves were constructed. At 24, 48, 72 and 96 h post-electroporation, mRNA of each group was detected by RT-PCR and real-time PCR, respectively. The PK activity was tested by ultraviolet spectrophotometry.
RESULTS
The growth curve showed that the growth of trophozoites was considerably depressed after 96 h post-electroporation. RT-PCR result displayed that the specific ribozyme mRNA was detected in group A from 24 h to 96 h post-electroporation. At 24 and 48 h after transfection, the PK mRNA level of group A decreased to 5% (5 +/- 0.17) and 8% (8 +/- 0.19) of the level in group C, respectively; and the PK activity of group A decreased to 32% (32 +/- 0.64) and 38% (38 +/- 0.65) of the level in group C.
CONCLUSION
PK mRNA expression in G. lamblia has been inhibited by specific hammerhead ribozyme.
目的
通过特异性锤头状核酶抑制蓝氏贾第鞭毛虫丙酮酸激酶（PK）mRNA的表达。
方法
将构建的针对PK mRNA的锤头状 - GCV载体（pGCV - PKH）电穿孔导入蓝氏贾第鞭毛虫滋养体（A组）。电穿孔后的滋养体（B组）和正常滋养体（C组）作为对照。分别在电穿孔后24、48、72和96小时收集每组的滋养体，计算滋养体浓度并绘制生长曲线。在电穿孔后24、48、72和96小时，分别通过RT - PCR和实时PCR检测每组的mRNA，通过紫外分光光度法检测PK活性。
结果
生长曲线显示电穿孔后96小时滋养体的生长明显受到抑制。RT - PCR结果显示，电穿孔后24小时至96小时在A组中检测到特异性核酶mRNA。转染后24小时和48小时，A组的PK mRNA水平分别降至C组水平的5%（5±0.17）和8%（8±0.19）；A组的PK活性分别降至C组水平的32%（32±0.64）和38%（38±0.65）。
结论
特异性锤头状核酶抑制了蓝氏贾第鞭毛虫中PK mRNA的表达。
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