摘要
目的:入神经生长因子包括β-神经生长因子、神经生长因子这两种活性形式,拟分别构建这两种活性形式的基因重组真核表达载体,为进一步的转基因实验作好前期准备。方法:实验于2001～2004年在青岛大学医学院附属医院中心实验室完成。①对象:健康志愿者5人,均对本实验知情同意。流产胎儿由青岛大学医学院妇产科提供,产妇均签署知情同意书,实验经医院医学伦理委员会批准。②实验方法:根据人β-神经生长因子与神经生长因子的全长基因序列设计合成特异性引物。抽取正常人外周血1.5 mL,提取基因组DNA后克隆出人β-神经生长因子基因片段。取胎儿海马部位脑组织100 mg,采用RT-PCR技术从胎儿脑组织中克隆出人神经生长因子基因片段。分别将纯化的β-神经生长因子产物与神经生长因子产物连接于真核表达载体pcDNA_4上,构建重组真核表达载体。转入JM109大肠杆菌中,扩增后小剂量质粒提取,酶切鉴定及计算机自动测序。结果:β-神经生长因子和神经生长因子的PCR产物琼脂糖凝胶电泳结果显示,在预期位置均有阳性条带。酶切鉴定结果及计算机自动测序均证实插入片段为目的基因片断。结论:实验分别从人外周血和胎脑海马组织中成功克隆获得β-神经生长因子与神经生长因子,并顺利构建这两种活性形式的重组真核表达载体pcDNA_4-β-NGF与pcDNA_4-NGF,为进一步将神经生长因子基因转入真核细胞并比较二者表达效率作好准备工作。
AIM： Human nerve growth factor is composed of two active kinds of β-nerve growth factor and nerve growth factor. This article aims to construct recombinant eukaryotic expression vector of two active kinds of human nerve growth factor to provide the scientific basis for gene therapy and clinical experiment.
METHODS： Experiments were performed at the Central Laboratory in Affiliated Hospital of Medical College of Qingdo University from 2001 to 2004. ①Five normal human （all healthy volunteers with agreement and understand of all the details about this project） were enrolled. Miscarried fetuses were provided by Department of Gynecology of Medical College of Qingdao University. Experiments were approved by hospital medical ethics committee. ②A pair of specific primers were designed and synthesized according to the total length gene sequence of β-nerve growth factor and nerve growth factor. 1.5 mL of peripheral blood was collected from normal persons. Human β-nerve growth factor gene fragment was cloned after extracting genome DNA. 100 mg of the brain tissue was obtained from the β-NGF gene was inserted into pcDNA4 vector to construct recombinant eukaryotic expression plasmid pcDNA4-β-NGF; which was propagated in Escherichia coil JM109, then extracted and purified followed by the sequencing and enzyme digestion analysis.
RESULTS： Positive bands appeared in the prospective place by gel electrophoresis; the inserted fragment was confirmed by sequencing and enzyme digestion analysis.
CONCLUSION： Two kinds of active human nerve growth factor are obtained from the human peripheral blood and the hippocampus of fetuses. The recombinant eukaryotic expression vector pcDNA4-β-NGF and pcDNA4-NGF are successfully constructed thus provide scientific basis for gene therapy and advanced clinical research.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第50期10121-10123,共3页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金(30572011)~~
