| 特异性锤头状核酶对蓝氏贾第鞭毛虫丙酮酸激酶mRNA的表达抑制 |
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| 作者姓名： | Feng XM Cao LJ Wang FY Zhang XC Lu SQ |
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| 作者单位： | 1 吉林医药学院病原生物教研室,吉林 132013;2 河北省儿童医院重症监护科,石家庄 050031;3 首都医科大学病原生物学系,北京 100069;4 吉林大学畜牧医学院,长春 130062 |
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| 摘 要： | 目的应用特异性锤头状核酶技术研究蓝氏贾第鞭毛虫丙酮酸激酶(pyruvate kinase)mRNA的表达抑制。方法用电击转染的方法将含有针对贾第虫丙酮酸激酶mRNA的核酶pGCV-PKH载体导入蓝氏贾第鞭毛虫滋养体细胞内(A组),同时设单纯电击转染滋养体(B组)和正常培养滋养体(C组)为对照。转染后24、48、72和96h收集各组虫体,计算滋养体浓度,绘制虫体生长曲线。提取虫体总RNA,分别采用RT-PCR和实时PCR方法检测转染后各组各时间段(24、48、72和96h)核酶mRNA和丙酮酸激酶mRNA相对含量的变化,用紫外分光光度法检测丙酮酸激酶活性。结果虫体生长曲线显示,转染96h,A组虫体的生长受到明显抑制。RT-PCR检测结果表明,A组在转染后24h即可在贾第虫滋养体细胞内检测到核酶RNA,其水平可持续至转染后96h。A组在电击转染后24和48h,丙酮酸激酶mRNA的表达量分别下降至C组的5%(5.00±0.17)和8%(8.00±0.19),相应的酶活性下降至C组的32%(32.00±0.64)和38%(38.00±0.65)。结论特异性锤头状核酶显著抑制了蓝氏贾第鞭毛虫丙酮酸激酶mRNA的表达。
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| 关 键 词： | 蓝氏贾第鞭毛虫 丙酮酸激酶 锤头状核酶 |
Inhibition of pyruvate kinase mRNA expression in Giardia lamblia by specific hammerhead ribozyme |
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| Feng XM,Cao LJ,Wang FY,Zhang XC,Lu SQ.Inhibition of pyruvate kinase mRNA expression in Giardia lamblia by specific hammerhead ribozyme[J].Chinese Journal of Parasitology and Parasitic Diseases,2010,28(4):257-260. |
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| Authors: | Feng Xian-min Cao Li-jing Wang Feng-yun Zhang Xi-chen Lu Si-qi |
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| Affiliation: | Department of Pathogen Biology, Jilin Medical College, Jiln 132013, China. |
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| Abstract: | 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. lamblia 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 realtime 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. |
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| Keywords: | Giardia lamblia Pyruvate kinase Giardia lamblia" target="_blank">Hammerhead ribozyme')">Giardia lamblia Pyruvate kinase Hammerhead ribozyme |
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