抗血小板衍生生长因子受体β亚单位核酶的体外制备与活性鉴定

Preparation and identification of activity of antiplatelet-derived growth factor receptor β subunit hammerhead ribozyme in vitro

目的 研究针对血小板衍生生长因子（PDGF）受体β亚单位基因的核酶的制备与体外切割活性鉴定。 方法 将大鼠PDGF受体β亚单位基因的PCR片段克隆于T载体T7启动子下游，32P标记的体外转录物作为靶RNA；设计并合成针对大鼠PDGF受体β亚单位胞外区的核酶，将核酶基因克隆于自我切割核酶载体P1.5的5′-cis核酶和3′-cis核酶之间，并进行32P标记的转录。核酶与靶RNA按一定比例和条件进行切割反应电泳，放射自显影。 结果 核酶制备正确，在最适条件下具有切割活性。其Km＝13.20nM，Kcat＝0.28min-1。最高切割效率达78.3%。 结论 体外制备的核酶具有良好的特异催化切割活性。

Objective To study the preparation and cleavage activity of ribozyme directed against platelet-derived growth factor (PDGF) receptor β subunit gene transcript in vitro. Methods PDGF receptor B subunit gene fragments were cloned into T-vector under the control of T7 promotor. 32P-labeled PDGF receptor β subunit transcripts as target-RNAs were transcribed in vitro and purified by PAGE. Ribozyme gene designed by computer targeting the extracellular domain was cloned into vector P1.5 between 5'-cis ribozyme and 3'-cis ribozyme. 32P-labeled ribozyme transcripts was gel-purified, incubated with target-RNAs at different conditions and autoradiographed after denaturing gel-electrophoresis. Results The preparation of ribozyme was correct. It was active at 37℃. The optimal temperature was 50℃, Km=13,20 nm, Kcat=0.28 min-1. The cleavage efficiency was up to 78.3%. Conclusions Ribozyme prepared in vitro possesses specific catalytic activity. [