真核表达质粒pcDNA3.1(＋)-h/mCD44v6的构建和鉴定  

Construction and determination of a recombinant eukaryotic plasmid pcDNA3.1(+)-h/mCD44v6

作　　者：许铁峰[1,2] 夏立平[1] 陈兴[2] 阎瑾琦[2] 于继云[2] 

机构地区：[1]海南医学院肿瘤研究所,附属医院肿瘤外科,海南海口570102 [2]军事医学科学院基础医学研究所疫苗工程研究室,北京100850

出　　处：《中国药理学通报》2009年第8期1111-1113,共3页Chinese Pharmacological Bulletin

基　　金：国家高技术研究发展计划(863计划)资助项目(No2001AA217131);海南省教育厅资助项目(NoHjkj2006-26);海南省自然科学基金重点项目(No061002);海南省自然科学基金项目(No806129)

摘　　要：目的构建含人/鼠嵌合粘附分子CD44拼接变异体6(chimeric human and mouse CD44 splice variant 6,h/mCD44v6)基因真核表达质粒pcDNA3.1(+)-h/mCD44v6,并进行表达。方法通过PCR扩增h/mCD44v6的全基因cDNA,将扩增的cDNA克隆至PGM-Teasy,测序后插入pcD-NA3.1(+)真核表达质粒,构建重组真核表达质粒pcD-NA3.1(+)-h/mCD44v6,经限制内切酶酶切分析及测序鉴定后,用脂质体转染B16细胞,通过RT-PCR扩增出B16/pcDNA3.1(+)-h/mCD44v6细胞株中h/mCD44v6全段基因cDNA。结果经4轮PCR,成功扩增出h/mCD44v6的cD-NA全长基因,成功构建了真核表达质粒pcDNA3.1(+)-h/mCD44v6,通过RT-PCR方法证实该质粒能在B16真核细胞中正确表达;建立了稳定的细胞株B16/pcDNA3.1(+)-h/mCD44v6。结论成功克隆和构建了h/mCD44v6的真核表达质粒pcDNA3.1(+)-h/mCD44v6,为进一步研究h/mCD44v6的新功能和免疫治疗奠定了基础。Aim To construct a eukaryotic plasmid containing the chimeric human and mouse CD44 splice variant 6 gene （h/ mCD44v6） and test the expression of the plasmid in the eukaryotic cell. Methods The full-length cDNA of h/mCD44v6 encoding gene was obtained by PCR. Then the cDNA was inserted into the eukaryotic expression vector pcDNA3.1 （ ＋ ） ,the resultant recombinant plasmid was confirmed by restriction endonuelease and sequencing, then designated as pcDNA3.1 （ ＋ ）-h/ mCD44v6. The recombinant eukaryotic plasmid pcDNA3.1 （ ＋ ）-h/mCD44v6 was transfected into eukaryotic cell B16 by LIPOFECTIN. RT-PCR was used to certify whether the purpose gene was correctly expressed in B16/pcDNA3.1 （ ＋ ）-h/ mCD44v6 cell. Results About 358 bp cDNA of h/mCD44v6 was obtained by four steps of overlapping PCR. The recombinant eukaryotic expression plasmid pcDNA3. 1 （ ＋ ）-h/mCD44v6 was successfully constructed and expressed in the B16 cells. Conclusion This recombinant pcDNA3. 1 （ ＋ ）-h/mCD44v6 plasmid will provide a basis for further study on the unknown function of h/mCD4 v6.

关 键 词：粘附分子CD44拼接变异体6 质粒 真核表达 细胞株 基因 克隆 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R392.11[医药卫生—基础医学]