摘要
目的研究针对大鼠PDGF受体β亚单位基因2个不同切割位点核酶的体外切割活性并加以比较。方法应用计算机对大鼠PDGF受体β亚单位mRNA二级结构进行模拟,设计针对PDGF受体β亚单位mRNA 4 5位CUU和2 5 2位CUU的锤头状(Hammerhead)核酶(Ribozyme)基因RZ1和RZ2 ,定点克隆于自我切割型核酶载体P1 5的5′cis核酶和3′- cis核酶之间;将大鼠PDGF受体β亚单位cDNA6 0 8bp的PCR片段克隆于pGEM T载体T7启动子下游,在T7启动子作用下分别体外转录成RZ1、RZ2和70 6nt的PDGF受体β亚单位mRNA ,比较RZ1和RZ2对70 6nt的PDGF受体β亚单位mRNA的切割活性的差异。结果RZ1在体外有较高的切割活性,而RZ2在体外无切割活性。结论提示在应用核酶技术进行基因治疗时,应选择有效的核酶切割位点,以达到有效的治疗目的。
To study and compare the cleavage activity of ribozymes directed against different sites of PDGF receptor β subunit gene in vitro. Methods The 608bp fragment of PDGF receptor β subunit cDNA were cloned into T-vector under the control of T7 promotor, named pPDGFR-β. Two ribozymes have been designed to cleave the CUU sequence at codon 45 and CUU sequence at codon 252 of PDGF receptor β subunit mRNA according to the analysis of the secondary structure of PDGF receptor β subunit mRNA with computer. The hammerhead ribozyme genes were cloned into vector P1.5 between 5′-cis ribozyme and 3′-cis ribozyme named pRZ1 and pRZ2 respectively. Results The pPDGFR-β as well as pRZ1 and pRZ2 were linearalized and transcribed with T7 promotor. The RZ1 showed high cleavage activity in vitro, but the RZ2 showed no cleavge activity under the same condition. Conclusion All these suggested that the cleavage site selection is an important factor which influences the cleavage activities of ribozymes.
出处
《基础医学与临床》
CSCD
北大核心
2005年第2期134-136,共3页
Basic and Clinical Medicine
基金
国家自然科学基金 (39870 30 3)
