摘要
目的:利用巢式逆转录-聚合酶链反应扩增的方法,从肌肉组织中扩增人骨形成蛋白2全长cDNA并构建真核表达载体系统。方法:实验于2003-10/2005-10在苏州大学基因工程教研室和北京大学第三医院骨科实验室完成。提取成人肌肉组织内的总RNA,设计内外两对引物以巢式逆转录-聚合酶链反应扩增方法分两次扩增出人骨形成蛋白2全长1188bp基因,经T-A克隆装入pUCM-T质粒载体内,测序验证后,将克隆质粒以Hind Ⅲ和Xba Ⅰ双酶切后与pcDNA3.0载体相连接,构建真核表达载体系统。结果:利用巢式逆转录-聚合酶链反应扩增方法能从成人肌肉组织内扩增出1188bp的人骨形成蛋白2全长cDNA基因,其测序结果显示与Genebank报道序列完全相符。将扩增序列双酶切后与pcDNA3.0载体相连接,经电泳验证,能构建人骨形成蛋白2全长基因的真核表达系统。结论:巢式逆转录-聚合酶链反应扩增方法能从成人肌肉组织内扩增出人骨形成蛋白2全长cDNA基因,并克隆构建真核表达载体系统,为下一步基因组织工程人工骨实验奠定基础。
AIM： To amplify the full-length cDNA gene of human bone morphogenetic protein-2 （hBMP2） and construct eukaryotic expression vector with nested reverse transcription-polymerase chain reaction （RT-PCR）.
METHODS： The experiment was performed at the Gene Engineering Lab, Soochow University and Laboratory of Department of Orthopedics, Third Hospital, Peking University from October 2003 to October 2005. Total RNA of some muscle tissues obtained from adult operation remnant. Two pairs of primers were designed. Using nested RT-PCR, 1 188 bp full-length hBMP2 gene was amplified from adult skeletal muscle and then cloned into pUCM-T vector by T-A. After double enzyme digesting with Hind 11I and Xba I , the gene was inserted into pcDNA3.0 vector to construct a eukaryotic expression system.
RESULTS： With the nested RT-PCR, 1 188 bp full-length hBMP2 gene was amplified from adult skeletal muscle, and then after the gene was validated by DNA agarose gel electrophoresis, it was digested by HindⅢ and Xba Ⅰ and cloned into pcDNA3.0 plasmid to construct eukaryotic expression system.
CONCLUSION： The 1 188 bp full-length hBMP2 gene can be amplified from adult skeletal muscle with nested RT-PCR to construct the eukaryotic expression system, which will provide a basis for tissue engineering artificial bone.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第14期2702-2704,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
