针对α_1 Ⅰ型及α_1 Ⅲ型前胶原基因第二外显子片段核酶的构建  

Construction of specific ribozymes against second exon of α_1 Ⅰ and α_1 Ⅲ procollagen genes

作　　者：朱家源[1] 朱斌[1] 徐兵[2] 张涛[1] 陈东[1] 苏爱云[1] 郭浩光[1] 唐斌[1] 

机构地区：[1]中山大学附属第一医院烧伤外科,广州510080 [2]第一军医大学南方医院

出　　处：《中华医学美学美容杂志》2004年第1期30-33,共4页Chinese Journal of Medical Aesthetics and Cosmetology

基　　金：国家自然科学基金 (3 9770 842 )

摘　　要：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 two ribozyme genes (gⅠand gⅢ) by using PCR according to the requirement of ribozyme's domains and the nucleotide sequences of α_1Ⅰand Ⅲ procollagen genes. Methods First we constructed two ribozyme genes (g, gⅢ) by using PCR, and then separately cloned into the downstream of T7 promoter of pGEM-T vector. Results The resulting recombinant plasmids (pT-gⅠ, pT-gⅢ) were verified by restriction enzymes, PCR and sequencing. Conclusion The expected two ribozyme genes are constructed successfully and this work affords the basis for the preparation of large amounts of ribozyme molecules and for the study on interaction between mRNA and ribozyme in vivo.

关 键 词：α1Ⅰ型前胶原基因 α1Ⅲ型前胶原基因 第二外显子 核酶 聚合酶链反应 

分 类 号：R346[医药卫生—基础医学]