HCVIRES特异性抑制性RNA二级结构模拟及自剪切转录载体构建

Structure modeling and construction recombinant plasmid of HCV IRES specific inhibitor RNA(IRNA)

目的 :构建可保证HCVIRES特异性抑制性RNA(IRNA)序列完整性的转录载体。 方法 :应用计算机软件模拟IRNA二级结构 ;体外化学合成IRNA及其互补体的cDNA ,以AatⅡ和XbaⅠ双酶切后克隆入具自剪切作用的顺式核酶载体pGEMRz中 ;应用酶切方法、PCR和测序法对重组载体进行三重鉴定 ;最后对重组体进行体外转录。 结果 :计算机模拟发现HCVIRES特异性IRNA二级结构包括两个环、一个茎状结构和一个膨出 ,在其序列两端加3～ 5个碱基不会改变其二级结构 ,而缺失 3～ 5个碱基却会明显改变其结构。经三重鉴定证实 ,目的序列被正确地克隆入质粒pGEMRz,并经体外转录得到约 70个碱基大小的IRNA。 结论 :本研究构建转录载体pGRz IRNA可保证IRNA序列的完整性。

Objectives:To construct transcript plasmid of HCV IRES specific inhibitor RNA which can provide correct secondary structure of IRNA. Methods:Using biosoftware to model the secondary structure of HCV IRES specific IRNA and in vitro synthesized cDNA of IRNA and its complementary RNA with XbaⅠ overhanging at 5′end and AatⅡ at 3′end,then cut with AatⅡ and XbaⅠ and placed in a tripartite ligation reaction with pGEMRz(cut with AatⅡ and XbaⅠ),which has two different cis ribozyme within the mutiplenzyme region. Then using enzyme cutting,PCR and sequencing to identify the recombinant plasmid. At last transcribing the vector in vitro . Results:Computer modeling of the secondary structure of IRNA and mutant IRNA using the free energy minimizing RNA structure 3.6 and WDNAsis indicated that the major structure of IRNA was a stem ,two loops and a bulge.Adding some base pares at two sides respectively or together never change the major structure of IRNA but losing some base pares at the two sides can change the major structure significantly.Identified through different way,the IRNA was proved to be correctly inserted into the plasmid pGEMRz.And through in vitro transcription about 70 bp RNA was generated. Conclusions:This study successively construct the recombinant plasmids of IRNA which can provide correct end of both sites through overhanging cis ribozyme at both sides.