摘要
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。
AIM To analyze in vitro self-cleavage activity of hepatitis delta virus ribozymes (HDV. Rz) with different length.METHODS Two plasmids pRz277A and pRz277B which contain the HDV. Rz cDNA were linearized with restriction endoncleases Xba Ⅰ, EcoR Ⅰ or BamH Ⅰ. Various genomic ribozymes (g. Rz) and antigenomic ribozymes (ag. Rz) were transcribed from their templates with T7 phage RNA polymerase and labeled with α-32 P-UTP. After reaction under certain conditions, these ribozymes were identified by 12% denatured polyacrylamide gel electrophoresis (PAGE) and autoradiography. RESULTS g.Rz24/100 which contain 24 nucleotides (nt) and 100nt at the 5' and 3' end of the cleavage site respectively and ag.Rz38/119 can almost be completely self-cleaved under proper conditions in vitro; g.Rz24/253 and ag. Rz38/ 239 also had self-cleavage activity but significantly lower than the former two ones, even when the temperature was up to 55℃ or different densities of deionized formamide were added.CONCLUSION HDV. Rz with appropriate sizes have high self-cleavage activity in vitro and these findings will contribute to the research of transcleavage activity of HDV Rz.
出处
《世界华人消化杂志》
CAS
2000年第1期39-41,共3页
World Chinese Journal of Digestology
基金
国家自然科学基金
No.39600031
关键词
丁型肝炎病毒
病毒核酶
自剪切活性
hepatitis D virus
virus ribozymes
self cleavage activity
