肾癌G250基因和hGM-CSF基因双顺反子表达质粒的构建及鉴定  

Construction and identification of bicistronic eukaryotic expression plasmid containing G250 of renal carcinoma and hGM-CSF

作　　者：李科[1] 李君武[2] 苏泽轩[1] 刘艳[2] 

机构地区：[1]暨南大学附属第一医院泌尿外科,广东广州510630 [2]暨南大学医学院微生物与免疫教研室,广东广州510632

出　　处：《中国现代医学杂志》2009年第8期1176-1179,1182,共5页China Journal of Modern Medicine

基　　金：广东省"十一五"医学重点专科基金[2008(粤卫)50号]

摘　　要：目的应用分子生物学技术,构建pIG250-GM双顺反子真核表达质粒并进行测序鉴定。方法从肾癌组织中提取总RNA,采用RTPCR技术扩增肾癌G250抗原的基因编码区序列,同时从质粒pORF-hGM-CSF中扩增出基因佐剂hGM-CSF,分别克隆到真核表达载体pIRES的多克隆位点A(MCSA)和B(MCSB)中,构建pIG250-GM真核表达质粒,并进行酶切鉴定。结果酶切鉴定提示插入的基因片段大小分别为1.44kμ和0.47kμ,测序分析表明克隆的G250和hGM-CSF序列与GenBank上公布序列完全一致。结论成功构建双顺反子表达pIGM-G250质粒,为研究G250-GMCSF基因负载的DC疫苗防治肾癌提供了必要的实验基础。[Objective] To construct and identify the bicistronic eukaryofic expression plasmid pIG250-GM by moleculal-biological methods, [Methods] The total RNA was extracted from RCC tissue; and G250 antigen eDNA was amplified by RT PCR, and then we used eDNA as template to amplify G250 gene and used plasmid pORF- hGM-CSF as template to amplify gene adjuvant hGM-CSF gene （granulocyte- maerophage colony-stimulating factor） by PCR, and then recombined them with the two multiple cloning sites （MCSA and MCSB） of plRES vector to construct a bicistronic eukaryofic expression plasmid pIG250-GM. [Results] Restriction enzyme digestion demonstrated inserted gene fragments were 1.44 kμ, and 0.47 kμ, DNA sequencing revealed that the cloned G250 and hGM-CSF sequences were consistent with the Gen Bank reported, and the target genes were correctly cloned into the vector. [Conclusion] The successful construction of a bicistronic eukaryofic expression vector containing G250 gene and gene adjuvant hGM-CSF, lays a foundation for researches on DNA vaccine and molecular biology in the therapist of renal carcinoma.

关 键 词：肾癌 G250 HGM-CSF 基因佐剂 

分 类 号：R737.11[医药卫生—肿瘤] Q78[医药卫生—临床医学]