真核表达质粒PIRES2-AcGFP-DNTβRⅡ的构建及其在COS-7细胞中的表达  

Costructio of eukaryotic expression vector PIRES2-AcGFP-DNTβRⅡ and expression of DNTβRⅡ in COS-7 cells

作　　者：杨波[1] 刘辉[1] 吴亮亮[1] 张丽娅[2] 焦顺昌[1] 

机构地区：[1]解放军总医院肿瘤内科,北京100853 [2]第二炮兵总医院干部病区,北京100088

出　　处：《现代肿瘤医学》2010年第4期643-646,共4页Journal of Modern Oncology

基　　金：军队十一五攻关项目(项目编号:06G106)

摘　　要：目的:通过oligo化学合成及PCR扩增法合成人显性抑制TGF-βⅡ型受体目的基因,并建立其真核表达载体。方法:根据Genebank数据库确定显性抑制TβRⅡ目的基因,利用化学合成法进行单链oligo的合成,PCR法将合成的oligo拼接成完整的序列,装入PMD18-T载体并转染感受态细胞DH5。经XhoI和Eco-RI酶切后连接至目的载体PIRES2-AcGFP中,转染COS-7细胞,对此质粒载体进行酶切电泳鉴定和DNA测序鉴定,用倒置荧光显微镜观察绿色荧光蛋白的表达,Western blot检测DNTβRⅡ表达。结果:合成DNA经测序验证与目的基因一致;PIRES2-AcGFP-DNTβRⅡ转染COS-7细胞,实现了DNTβRⅡ目的基因在COS-7细胞中的体外转染及瞬时表达。结论:采用oligo化学合成及PCR扩增法成功合成人显性抑制TGF-βⅡ型目的基因并构建了其真核表达载体PIRES2-AcGFP-DNTβRⅡ,为进一步研究过继性免疫细胞治疗奠定了基础。Objective：To synthesize the gene of human dominant-negative transforming growth factor beta receptor Ⅱ（DNTβRⅡ） by means of oligo chemic synthesis and PCR amplification,and construct its eukaryotic expression vector.Methods： According to the Genebank Database,the sequence of target gene DNTβRⅡ was determined.The single chain oligo was synthesized chemically and then was ligated into full length gene by PCR.The synthesized gene was cloned into plasmid PMD18-T and transferred into competent cells DH5α,then was inserted into the corresponding restriction site on eukaryotic expression vector PIRES2-AcGFP.After transferring into COS-7 cells,expression of green fluorescent protein was identified by inverted fluorescent microscope and DNTβRⅡ protein was confirmed by Western blotting analysis.Results： The synthesized fragment was consistent with the target gene DNTβRⅡby sequence analysis.After the recombinant expression plasmid transfected into COS-7 cells,DNTβRⅡ was instantaneously transfected and expressed in vitro successfully.Conclusion： Full length gene of DNTβRⅡcan be synthesized by means of oligo chemic synthesis and PCR amplification,and the construction of its eukaryotic expression vector PIRES2-AcGFP-DNTβRⅡmay provide basis for further study of anti-tumor immunotherapy by adoptive immune cell therapy.

关 键 词：转化生长因子 受体 全基因合成 真核表达 

分 类 号：R730[医药卫生—肿瘤]