前列腺癌特异突变DNA聚合酶β真核表达载体构建及鉴定  

The construction and identification of recombinant eukaryotic expression vector containing specific mutant DNA polβ gene derived from prostate cancer

作　　者：王明臣[1] 宋宇[1] 张善锋[1] 付蕾[2] 文明[3] 赵勤[1] 金国平[1] 赵国强[4] 

机构地区：[1]郑州大学基础医学院生物化学与分子生物学教研室,河南郑州450052 [2]郑州大学药学院生物化学与分子生物学教研室 [3]河南武警总队医院 [4]微生物与免疫学教研室

出　　处：《中国老年学杂志》2009年第14期1758-1759,共2页Chinese Journal of Gerontology

基　　金：河南省科技厅自然科学基金(0611043400)

摘　　要：目的构建前列腺癌(PCa)特异突变DNA聚合酶β(polβ)真核表达载体。方法提取有特异DNApolβ突变的PCa组织的总RNA,通用引物逆转录成cDNA;用设计带有接头的DNA polβ全基因引物,PCR扩增PCa组织中呈现的突变型DNApolβ全基因;构建T-A克隆并进行重组体的筛选鉴定;亚克隆入表达载体pcDNA3.1并进行重组体的筛选和鉴定。结果PCa特异突变DN Apolβ基因扩增选择出P4(M1-polβ)和P5(M2-polβ)两个标本,构建重组有特定突变位点的polβ目的基因的pcDNA3.1真核表达载体,经PCR扩增获得2个阳性集落。结论经鉴定成功构建2例PCa特异突变DNApolβ真核表达载体(pcDNA3.1-M1 and pcDNA3.1-M2)。Objective To construct and identify the recombinant eukaryotic expression vector containing specific mutant DNA polβ gene derived from prostate cancer. Methods Total RNA was extracted from prostate cancer tissues with specific DNA polβ mutation and reversely transcripted to cDNA by universal primer. Utilizing primer with adapter design, the full length DNA polβ gene with specific mutation sites was amplificated by PCR. T-A clone was constructed and the recombinant was screened and identified. Full length DNA polβ gene with specific mutation sites was then subcloncd into expression vector pcDNA3.1 and the recombinant was screened and identified. Results P4 （M1-polβ） and P5 （M2-polβ） were selected. Eukaryotic expression vector pcDNA3.1 containing specific mutant DNA polβ gene was constructed and recombined and two positive colony were acquired by PCR. Conclusions Two eukaryotic expression vectors containing specific mutant DNA poll3 derived with prostate cancer （pcDNA3. 1-M1 and pcDNA3, 1-M2） are successfully constructed.

关 键 词：前列腺癌 DNA聚合酶Β 基因突变 真核表达载体 

分 类 号：R735[医药卫生—肿瘤]