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

Inhibition of Pyruvate Kinase mRNA Expression in Giardia lamblia by Specific Hammerhead Ribozyme

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。

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 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.