抗bcr/abl mRNA特异性核酶作用于靶基因的细胞内外切割实验

Cleavage Effects of Specific Ribozyme on bcr/abl mRNA in Vitro and in Vivo

目的 将已构建的具有抗慢性粒细胞性白血病(CML)融合基因bcr/abl mRNA的含核酶载体,进行细胞内外实验,了解抗bcr/abl mRNA核酶的切割效应。方法 含有抗靶基因的真核表达载体pAVGS4经酶切后,其中抗CML核酶M1-GS RNA的DNA序列被构建到pUC19上得到pGS210,并经酶切和测序鉴定;合成bcr/abl融合位点前后的一段靶序列,克隆到pGEM-7zf(+)上得到p bcr-abl经测序鉴定,将pGS210和p bcr-abl进行体外转录,获得 M1-GS RNA及其作用底物,将两者混合、孵育,通过放射自显影术检测切割的效果;将pAVGS4转染到CML细胞株K652细胞内,通过RT-PCR方法了解核酶对于目标 RNA的作用效应。结果 p bcr-abl经酶切电泳及测序证实载体中含有目标序列,Ml-GS RNA作用于底物后,放射自显影术检测显示底物 RNA被有效切割。pAVGS4转染到K562细胞48和72h后,RT-PCR显示bcr/abl mRNA被有效切割。结论 pAVGS4可以有效地切割K562细胞内的bcr/abl mRNA,预期可作为CML分子靶向治疗的工具。

Purpose: To determine the effects of M1-GS RNA on bcr/abl mRNA in vitro and in vivo. Methods: Eukaryotic expression expression vector pAVGS4 containing M1-GS RNA was digested by EcoRI and SaU and M1-GS RNA was subcloned pUC19(p S210). The part of bcr/abl sequence was subcloned to pGEM-7fa(+) (p bcr-abl). Restriction enzyme analysis and DNA sequencing were used to identify the plasmids. Both plasmids were transcribed in vitro and mixed. Autoradiography was used to determine effects of the ribozyme on the substrate. Then pAVGS4 was transfected into K562 by X-tremeGENE Q2. Fotry-eight and seventy-two hours after transfection, the cells were collected and total RNA was extracted for evaluating the enzyme effectiveness by RT-PCR. Results: Restriction enzyme analysis and sequencing showed that the M1-GS RNA in the plasmids was identical with the template. The eukaryotic expression vector was successfully conducted. M1-GS RNA could effectively cleave the artificial substrates. And also, it showed by RT-PCR that bcr/abl mRNA in K562 cells were cleaved 48 hand 72 hr after transfection. Conclusions: pAVGS4 may be a useful tool for molecular target therapy in CML.