摘要
目的构建PLEGFP—BDNF真核表达重组逆转录病毒,为进一步在细胞中表达及移植治疗作准备。方法按照hBDNF基因全长编码序列设计合成引物,从人基因组DNA中扩增出744bp的hBDNF基因片段,插入到PMD18-T载体上,转化DH5α大肠杆菌菌株,获得PMD18T—BDNF克隆,对该克隆进行限制性酶切分析和DNA序列测定;从阳性克隆中获取hBDNF全长编码片段,与真核表达载体PLEGFP质粒连接,构建PLEGFP—BDNF真核表达重组质粒。重组质粒转化大肠杆菌JM109,再经氨苄LB培养基筛选,酶切,PCR与测序鉴定。结果PLEGFP—BDNF重组逆转录病毒构建成功。结论hBDNF在大肠杆菌中的成功表达在转基因治疗CNS病变方面具有潜在应用价值。
Objective Constructing the recombinant retrovirus vector PLEGFP - BDNF to prepare further expression and transrection in cells for transplantation Methods The preparation of specific for full - length BDNF coding sequence was designed and synthesized. The BDNF coding sequence was directly amplified from human genomic DNA and which was transformed into the host cells E - coli DH 5a to obtain the positive clone PMD 1ST - BDNF. The restriction enzyme analysis and DNA sequence detection confirmed that the inserted fragment of clone is the full - length BDNF coding sequence. The hBDNF DNA fragment was recovered from the clone and ligated with eukaryonic expression vector PLEGFP to construct the recombinant expression plasmid PLEGFP - BDNF. The E - coli JM 109 transformed with PLEGFP - BDNF was induced. The positive clone was screened on LB plates containing ampicillin and identified by restrictive enzymes digestion PCR amplification and DNA sequence detection. Results PLEGFP - BDNF was constructed successfully. Conclusios The successful expression of hBDNF in Escherichia coli has the potential application value in the transgenic therapy of CNS disease.
出处
《中国骨肿瘤骨病》
2005年第4期224-227,共4页
Chinse Journal Of Bone Tumor And Bone Disease
基金
云南省自然科学基金资助项目(2002C0070M)
