人胎脑NT-4基因的体外构建  

The Construction of NT-4 Gene of Human Embryonic Brain in Vitro

作　　者：李劲涛[1] 朱兴宝[2] 徐丹[1] 王廷华[1] 张华堂[3] 刘佳[1] 

机构地区：[1]昆明医学院神经科学研究所,云南昆明650031 [2]成都军区昆明总医院神经外科,云南昆明650032 [3]中国科学院昆明动物研究所,云南昆明650223

出　　处：《昆明医学院学报》2008年第4期65-70,共6页Journal of Kunming Medical College

基　　金：云南省教育厅科研基金重点项目(07C10388)

摘　　要：目的体外构建人神经生长因子-4(neurotrophin-4,NT-4)与质粒载体pEGFP-N1(卡那霉素抗性)的重组体,为后续的转基因移植治疗脊髓损伤创造条件.方法用Trizol法提取人胎脑组织总RNA,经RT-PCR扩增,EorRⅠ和BamHⅠ核酸内切酶酶切电泳纯化获得人神经营养因子-4(NT-4)DNA.用含质粒载体pEGFP-N1DNA的大肠杆菌接种经划板,挑选菌落、振荡培养,并用碱裂解法提取质粒载体pEGFP-N1DNA,经酶切、电泳纯化后,通过连接、转化感受态细胞,DNA小量提取法获得NT-4目的基因与质粒载体pEGFP-N1DNA的重组体,经DNA测序证实.结果用上述方法成功获得NT-4-pEGFP-N1重组体.结论此方法是一种体外构建人胎脑NT-4基因的成熟、稳定而有效的方法,可为后续的神经干细胞转染NT-4基因移植治疗神经系统损伤创造条件.Objective To Construct the recombinant of NT-4 gene of embryonic human brain and the vector of pEGFP-N1 in vitro to provide foundation for the transgenic therapy of spinal cord injury （SCI）. Methods By Using Trizol method to distill RNA from the brain tissues of human embryo, through RT-PCR and purification by electrophoresis we got the NT-4 DNA, verified by EorR Ⅰ and BamH Ⅰ digestion and DNA concentration detecting. By surge culture of the E.eoli containing the DNA of the the pEGFP-N1 vector and distilling of it by plasmid Miniprep Kit Ⅰ Protocol, we got the DNA of the pEGFP-N1 vector. After purification by electrophoresis, the protocol of link follow up translation of the E.coli competent cells JM109, and distilling of the DNA of recombinant of NT-4 and pEGFP-N1 vector by plasmid Miniprep Kit Ⅰ Protocol, we got the DNA of recombinant of NT-4 and pEGFP-N1, confirmed by enzyme digestion, electrophoresis and DNA sequence. Results We got the DNA of recombinant of NT-4 and pEGFP-N1 vector successfully. Conclusion Our method is a mature, stable and effective method to construct NT-4 gene of human brain in vitro which deserved to be applied in the research of gene therapy of spinal cord injury.

关 键 词：人胎脑 神经营养因子-4 基因构建 体外 

分 类 号：R651[医药卫生—外科学]