人叉头转录因子O亚型3基因重组表达质粒的构建及鉴定  

Construction and identification of recombinant expression vector for human forkhead box class O3

作　　者：段志青[1] 段云青[2] 雷焕贵[2] 胡凝珠[1] 罗婷[1] 李虹娟 王霄[1] 胡云章[1] 

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所疫苗研究室云南省重大传染病疫苗研发重点实验室,云南昆明650118 [2]山西农业大学文理学院,山西太谷030801 [3]昆明医科大学生物所,云南昆明650500

出　　处：《中国生物制品学杂志》2014年第3期316-319,324,共5页Chinese Journal of Biologicals

基　　金：国家高技术研究发展计划(863计划)"新型疫苗佐剂的研发及其应用研究"(2012AA02A406);云南省创新团队"中国医学科学院医学生物学研究所新型疫苗佐剂应用研究省创新团队"(2011CI140)

摘　　要：目的构建人叉头转录因子O亚型3(forkhead box class O3,FOXO3)基因重组表达质粒,并检测其在人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)中的表达。方法利用3对引物从人cDNA中分别扩增大小为382、829和891 bp的3个目的片段,胶回收后进行拼接,获得hFOXO3的CDS片段,与pcDNA3.1(+)载体连接,构建重组表达质粒pcDNA3.1(+)-hFOXO3,转染人PBMC,采用Real-time PCR和Western blot分别检测hFOXO3基因mRNA水平及蛋白表达水平。结果重组表达质粒经双酶切及测序证实构建正确;转染PBMC后,pcDNA3.1(+)-hFOXO3组hFOXO3基因mRNA的水平分别为转染试剂对照组和pcDNA3.1(+)组的2 599.7和2 377.8倍,且差异均有统计学意义(P<0.001);pcDNA3.1(+)-hFOXO3组hFOXO3蛋白的表达水平分别为转染试剂对照组和pcDNA3.1(+)组的2和1.8倍,且差异均有统计学意义(P<0.01);而转染试剂对照组和pcDNA3.1(+)组间hFOXO3基因mRNA水平和蛋白的表达水平差异均无统计学意义(P>0.05)。结论成功构建了pcDNA3.1(+)-hFOXO3重组表达质粒,其在人PBMC中能进行hFOXO3基因mRNA的转录及蛋白的表达,为进一步研究hFOXO3的生物学功能及其作用机制奠定了基础。Objective To construct the recombinant expression vector for human forkhead box class 03 （hFOXO3）, and determine its expression levels in human peripheral blood mononuclear cells （PBMCs）. Methods Three target gene fragments, at lengths of 382, 829 and 891 bp, were amplified from human eDNA by PCR using three pairs of primers, respectively, then recovered with gel and spliced. The obtained CDS fragment of hFOXO3 was inserted into vector pcDNA3. 1 （＋）. PBMCs were transfected with the constructed recombinant p]asmid pcDNA3. 1 （＋）-hFOXO3 and determined for mRNA transcription level of hFOXO3 by Real-time PCR, while the protein expression level by Western blot. Results Restriction analysis and sequencing proved that recombinant plasmid pcDNA3, l （＋）-hFOXO3 was constructed correctly. The FOXO3 mRNA expression level in PBMCs transfected with plasmid pcDNA3. I（＋）-hFOXO3 were 2 599. 7 and 2 377.8 times of those in transfection reagent and pcDNA3. 1 （＋） control groups （each P 〈 O. 001 ）, while the protein expression level were 2 and 1.8 times, respectively （each P 〈 0. 01 ）, respectively. However, neither mRNA nor protein expression level showed significant difference in transfection reagent and peDNA3, l（＋） control groups （each P 〉 0. 05）. Conclusion Recombinant plasmid pcDNA3. 1 （＋）-hFOXO3 was successfully constructed, and hFOXO3 was expressed in human PBMCs at both mRNA and protein levels, which laid a foundation of further study on the biological function and action mechanism of hFOXO3.

关 键 词：人叉头转录因子O亚型3基因 原核细胞 基因表达 

分 类 号：Q786[生物学—分子生物学]