摘要
目的:构建雄激素受体基因第一外显子区CAG重复序列缺失的突变重组体,观察CAG重复序列的有/无对雄激素受体转录及表达的调控作用。方法:实验于2006-05/2007-05在解放军总医院老年医学研究所完成。按照重叠延伸反应的原理扩增CAG重复序列两端的基因片段,再将两片段混合,在加入一个引物的情况下进行8个PCR循环以有效完成重叠延伸,然后再加入另一引物进行22个循环,完成目的片段指数扩增,将目的片段克隆至真核表达载体PAR-IRES2-EGFP,转染HEK293细胞,采用Western blot法检测HEK293细胞雄激素受体蛋白。结果:构建的CAG重复序列缺失的突变重组体经酶切鉴定和DNA测序证实构建正确,转染真核细胞可检测到雄激素受体基因的表达。结论:重叠延伸PCR是进行体外基因拼接、体外缺失突变的简单、快速的基因重组技术;雄激素受体基因第一外显子区CAG重复序列缺失的突变重组体的成功构建可为今后的相关研究提供实验基础。
AIM： To construct mutant recombinant plasmid with deletion of CAG repeats in exon 1 of human androgen receptor （AR） gene, and to observe the transcription and expression of AR with CAG repeats or not. METHODS： The experiment was conducted in the Institute of Geriatric Medicine, General Hospital of Chinese PLA between May 2006 and May 2007. Two gene fragments at each end of the deleted gene were obtained by overlap extension （OE）-PCR method and were mixed together for 8 PCR cycles with one primer to achieve effective overlapping, then the other primer was added for 22 PCR cycles for amplification of the desired gene. The desired fragments were cloned into eukaryotic vector PAR-IRES2-EGFP, then the mutant recombinant was transfected into HEK293 cells. The protein of androgen receptor was detected by Western blot. RESULTS： The constructed mutant recombinant with CAG repeats deletion was confirmed using restriction enzymes and DNA sequencing. Expression of AR was detected in HEK293 cells. CONCLUSION： OE-PCR is a simple and volant gene recombination technique for gene splicing and deletion mutation in vitro The successful construction of the mutant recombinant with deletion of CAG repeats in exon 1 of human androgen receptor gene provides the basis for further related study.
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第37期7389-7392,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
军队十一五面上课题(06G105)~~
