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目的分析不同长度丁型肝炎病毒核酶的体外自剪切活性。方法用XbaⅠ,EcoRⅠ或BamHⅠ将含有HDV.Rz序列的pRz277正向质粒(pRz277A)和pRz277反向质粒(pRz277B)线性化,以α-~(32)p-UTP标志和T7噬菌体RNA聚合酶转录出不同长度的基因组核酶(g.Rz)和抗基因组(即复制中间体)核酶(ag.Rz),在一定条件下进行自剪切反应,然后作聚丙烯酰胺凝胶电泳(PAGE)和放射自显影。结果 g.Rz24/100(自剪切位点5和3端分别含24nt和100nt,下同)、ag.Rz38/119在适当条件下绝大部分发生自剪切。g.Rz24/253和ag.Rz38/239也具有一定自剪切活性,但显著低于前两者,即使温度升至55℃或加入去离子甲酰胺也是如此。结论适宜长度的HDV.Rz在体外具有较高的自剪切活性。这些发现有助于我们下一步研究HDV.Rz的反式剪切作用。
Objective To analyze the in vitro self-shearing activity of different lengths of hepatitis D virus ribozyme. Methods The pRz277 positive plasmid (pRz277A) and the pRz277 inverted plasmid (pRz277B) containing the HDV.Rz sequence were linearized with XbaI, EcoRI or BamHI and transcribed with the alpha-32P-UTP marker and T7 phage RNA polymerase Different lengths of genomic ribozyme (g.Rz) and anti-genomic (ie, replicating intermediate) ribozyme (ag.Rz) were subjected to self-shearing reaction under certain conditions followed by polyacrylamide gel electrophoresis (PAGE) And autoradiography. Results g.Rz24 / 100 (from the cleavage site 5 and 3, respectively, containing 24nt and 100nt, the same below), ag.Rz38 / 119 under the proper conditions, most of the occurrence of self-shear. The Rz24 / 253 and ag.Rz38 / 239 also have some self-shearing activity, but are significantly lower than the former two, even when the temperature is raised to 55 ° C or deionized formamide is added. Conclusion The suitable length of HDV.Rz has high self-shear activity in vitro. These findings will help us to study the trans-shearing of HDV.Rz in the next step.