SD大鼠MSX-2基因真核表达载体的构建与功能鉴定  

Construction and functional identification of eukaryotic expression vector carrying Sprague-Dawley rat MSX-2 gene

作　　者：杨娴娴[1] 张梅[2] 颜召文[2] 张如鸿[1] 穆雄铮[1] 

机构地区：[1]上海交通大学医学院附属第九人民医院整复外科,200011 [2]上海交通大学医学院基础医学院病理学教研室

出　　处：《中华整形外科杂志》2008年第1期58-62,共5页Chinese Journal of Plastic Surgery

摘　　要：目的构建SD大鼠颅颌面发育相关基因MSX-2与PcDNA3．1融合的高效真核表达载体，为研究MSX-2基因的功能奠定基础。方法根据SD大鼠MSX-2基因的核苷酸序列，设计并合成引物，采用PCR技术，扩增编码MSX-2基因，TA克隆到PMD18-T载体上，再亚克隆到真核表达载体PcDNA3．1的BamHI和Xhol位点。对该重组体进行酶切鉴定，以及测序验证。重组体转染HEK293细胞，RT-PCR和Western blot检测重组MSX-2基因的RNA和蛋白表达情况。结果重组真核表达载体PcDNA3．1-MSX-2经PCR扩增、酶切鉴定均显示有476bp左右的特异性基因片断，证明MSX-2基因正向插入真核表达载体中；碱基序列的测定证明重组质粒中含有SD大鼠的MSX-2基因序列。重组体转染HEK293细胞，RT-PCR和Western blot可检测到重组MSX-2基因的mRNA和蛋白的特异表达。结论成功构建SD大鼠MSX-2基因的高效真核表达载体，为进一步研究MSX-2基因的功能及基因治疗奠定了基础。Objective To construct a high effective eukaryotic expressing plasmid PcDNA 3.1-MSX-2 encoding Sprague-Dawley rat MSX-2 gene for the further study of MSX-2 gene function. Methods The full length SD rat MSX-2 gene was amplified by PCR,and the full length DNA was inserted in the PMD18-T vector. It was isolated by restriction enzyme digest with BamHI and Xhol, then ligated into the cloning site of the PcDNA3.1 expression plasmid. The positive recombinant was identified by PCR analysis, restriction endonudease analysis and sequence analysis. Expression of RNA and protein was detected by RT-PCR and Western blot analysis in PcDNA3.1-MSX-2 transfected HEK293 cells. Results Sequence analysis and restriction endonudease analysis of PcDNA3.1-MSX-2 demonstrated that the position and size of MSX-2 cDNA insertion were consistent with the design. RT-PCR and Western blot analysis showed specific expression of mRNA and protein of MSX-2 in the transfected HEK293 cells. Conclusions The high effective eukaryotic expression plasmid PcDNA3.1-MSX-2 encoding Sprague-Dawley Rat MSX-2 gene which is related to craniofacial development can be successfully reconstructed. It may serve as the basis for the further study of MSX-2 gene function.

关 键 词：颅颌面发育 MSX-2基因 真核表达载体 构建 功能鉴定 SD大鼠 基因治疗 

分 类 号：R686[医药卫生—骨科学]