人脑源性神经营养因子成熟肽基因克隆及表达

【作者】 张虎； 杨吉成； 盛伟华； 缪竞诚； 李丽娥； 王金志； 白艳艳； 谢宇锋；

【机构】 苏州大学医学院基础医学系细胞与分子生物学教研室；

【摘要】 目的:构建表达人脑源性神经营养因子的基因工程菌。方法:人工合成一对含EcoRI 和BamHI 限制性酶切位点的特异引物,以健康成人末梢白细胞基因组DNA 作为模板。应用PCR 技术扩增hBDNF 成熟肽基因,用A-T 克隆法与pUCm-T 载体连接,pUCm-T-BDNF克隆片段再定向插入原核表达载体PBV220,构建的重组体PBV220-BDNF 转化大肠杆菌DH5a 经热诱导表达。结果:经DNA 测序和限制性酶切确证重组人载体片段为目的基因的核苷酸序列,并成功表达蛋白。结论:本研究成功地克隆出haBDNF 基因编码序列并在大肠杆菌中获得稳定表达,为今后的生物学活性和神经系统的基因治疗奠定了基础更多还原

【Abstract】 Objective:TO construct the genetically engineered bacterium for humanbrain-derived neurotrophic factor Methods:A pair of specific primers including the cleavage siteof restriction endonuclease EcoRI and BamHI has been synthesized,from the genomic DNA Byusing PCR the BDNF gene sequence was directly amplified from the genomic DNA isolated fromthe human leucocytes.,the fragment of resulting gene was then cloned into Pucm-T vector.Theinsert fragment from the recombinant pUCm-T-BDNF was directionally ligated with lineariedPBV220.DH5a was transformed with the expression recombinant PBV220-BDNF and induced byheat Result:The cloned Gene was identified as the sequence encoding hBDNF by DNAsequencing and sequencing restriction anaylsis.SDS-PAGE revealed the cloned gene expressedsuccessfully Conclusion:The clone containing the sequence encoding human BDNF is obtainedand expressed in DHSa.s fight.This study provides an approach for the study of activity and thegene treatment for the nervous system diseases.更多还原