人PRMT1真核表达载体的构建及鉴定  

Construction and identification of human PRMT1 gene eukaryotic vector

作　　者：李晨燕[1,2] 孙青竹[1] 杨旭东[1] 钟波[1] 韩燕[1] 吕社民[1] 

机构地区：[1]西安交通大学医学院遗传与分子生物学系,陕西西安710061 [2]西安市卫生学校生物化学教研组,陕西西安710054

出　　处：《细胞与分子免疫学杂志》2012年第4期384-387,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金资助项目(81070030;81170017);西安交通大学2011年度基本科研业务费(xjj2011021)

摘　　要：目的:构建人PRMT1基因真核表达载体,并进行表达检测及鉴定。方法:采用RT-PCR技术扩增人PRMT1全长cDNA;经过双酶切、连接等反应,构建pcDNA3.1(+)-PRMT1真核表达载体,并转化DH5α感受态细胞;用含氨苄青霉素的LB培养基筛选阳性克隆;经菌液PCR及测序鉴定重组质粒;瞬时转染A549细胞,采用实时定量PCR检测重组质粒的表达水平及PRMT1对eotaxin-1和ccr-3表达的影响。通过Western blot从蛋白水平检测重组质粒在真核细胞内的表达。结果:RT-PCR扩增的人PRMT1全长cDNA为1136 bp;所筛选出的pcDNA3.1(+)-PRMT1重组载体中插入片段与NCBI GenBank文库中人PRMT1 cDNA的序列完全一致;实时定量PCR和Western blot检测证实重组质粒在A549细胞内可高效表达;并且PRMT1与eotaxin-1和ccr-3的表达呈正相关性。结论:成功构建了pcDNA3.1(+)-PRMT1重组载体,为进一步研究PRMT1基因的作用机制奠定基础。AIM： To construct the eukaryotic recombinant expression plasmid of pcDNA3. 1 （ ＋ ）-PRMT1. METHODS： Human PRMT1 cDNA was amplified by reverse transcription polymerase chain reaction （RT-PCR）. After digested by BamH Ⅰ, Hind Ⅲ and ligation, PRMT1 was inserted into pcDNA3. 1 （ ＋ ） eukaryotic expression vector. The positive colonies were screened and identified by PCR and sequencing, pcDNA3. 1 （ ＋ ）- PRMT1 plasmid was then transfected into the cultured A549 cell line with LipofectamineTM 2000. Realtime-PCR and Western blot were used to detect the mRNA and protein expression of PRMT1 respectively. RESULTS： The PRMT1 cDNA was successfully amplified, and pcDNA3.1 （ ＋ ）- PRMT1 were constructed. The inserted sequence in pcDNA3.1 （ ＋ ）-PRMT1 was the same as the sequence of PRMT1 cDNA published in NCBI GenBank. Further, Realtime PCR and Western blot results validated the recombinant plasmid expressed in A549 cell line efficiently. CONCLUSION： pcDNA3. 1 （ ＋ ）-PRMT1 recombinant was successfully constructed.

关 键 词：PRMT1 真核表达载体 基因克隆 A549细胞 

分 类 号：Q78[生物学—分子生物学]