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目的 构建出针对α1Ⅰ型及α1Ⅲ型前胶原基因第 2外显子 (Exon 2 )片段的核酶。方法 根据α1Ⅰ型及α1Ⅲ型前胶原基因的碱基序列以及锤头型核酶 (ribozyme)的结构域要求 ,利用聚合酶链反应 (PCR)分别构建了针对各自的Exon 2区域的核酶基因 ,并克隆到T载体 (pGEM Tvector)T7启动子下游。结果 分别以引物对PⅠL/PⅠR及PⅢL/PⅢR进行PCR扩增 ,经琼脂糖凝胶电泳各得到约6 2bp的DNA片段 ,与预期的核酶基因大小一致。结论 经PCR限制性内切酶酶切及序列测定分析 ,证实为所设计的插入正确的核酶基因 ,从而为大量制备核酶及其体外切割实验和胞内与mRNA相互作用的研究打下了基础。
Objective To construct a ribozyme targeting the exon 2 fragment of α1Ⅰ and α1Ⅲ procollagen genes. Methods According to the nucleotide sequences of α1Ⅰ and α1Ⅲ procollagen genes and the domain requirements of hammerhead ribozyme, the ribozyme gene was constructed for each Exon 2 region by polymerase chain reaction (PCR) And cloned downstream of the T7 promoter (pGEM Tvector). Results PⅠL / PⅠR and PⅢL / PⅢR were amplified by PCR. The DNA fragment of about 6 2bp was obtained by agarose gel electrophoresis, which was consistent with the expected ribozyme gene size. Conclusion The results of PCR restriction endonuclease digestion and sequence analysis confirmed that the inserted ribozyme gene was designed, which laid the foundation for the mass production of ribozyme and in vitro cleavage experiments and the interaction between intracellular and mRNA .