腺病毒穿梭质粒pShuttle CMV-^+VEGF_(121)-IRES-hrGFP-1的构建和鉴定  

The Construction and Appraisal of pShuttle CMV-^+VEGF_(121)-IRES-hrGFP-1

作　　者：胡亮[1] 李谌[1] 刘丹平[1] 

机构地区：[1]辽宁医学院附属第一医院骨科,辽宁锦州121001

出　　处：《辽宁医学院学报》2009年第6期488-490,共3页Journal of Liaoning Medical University (LNMU) Bimonthly

基　　金：辽宁省教育厅创新团队项目;编号:2007T109

摘　　要：目的构建腺病毒穿梭质粒pShuttle CMV-^+VEGF_(121)-IRES-hrGFP-1,为构建表达具有抗原表位标记的血管内皮生长因子121(vascular endothelial growth factor 121,VEGF_(121)),并同时表达绿色荧光蛋白(green fluorescent protein,GFP)报告分子的腺病毒真核细胞表达载体打下基础。方法对目的基因供体质粒pTG19T-VEGF_(121)携带的VEGF_(121)基因测序和序列内部存在的限制性内切酶识别位点进行分析,利用PCR(Polymerase Chain Reaction,PCR)技术对pTG19T-VEGF_(121)携带的VEGF_(121)基因突变,以去除翻译终止密码子后的基因序列并在序列前后分别添加新的NotI和XhoI酶切位点。将突变后的VEGF_(121)基因(^+VEGF_(121))定向连入腺病毒穿梭质粒pShuttle CMV-IRES-hrGFP-1,通过电泳和测序鉴定获得的重组质粒。结果重组质粒经电泳和测序鉴定为正确克隆。结论成功构建了pShuttle CMV-^+VEGF_(121)-IRES-hrGFP-1。Objective To clone vascular endothelial growth factor 121（VEGF_121） gene into the adenovirus shuttle plasmid pShuttleCMV-IRFS-hrGFP-1,preparing for construction of a novel recombinant adenovirus vector expressing the VEGF_（121） fused to FLAG epitope and humanized Renilla reniformis green fluorescent protein 1（hrGFP-1） as a reporter on the same transcript.Methods The VEGF_（121） gene contained in the plasimid of pTG19T-VEGF_（121） was sequenced,and the profile of restriction endonuclease sits existing in the sequence was analysed.Then the translation stop codon TAG was removed.Meanwhile,Notl and Xho I restriction sites were added before the start codon and after the 3＇end of the mutant through polymeras chain reaction（PCR） mutagenesis technology. The mutant of VEGF_（121） gene（~＋VEGF_（121） gene） was ligated into the multiple cloning sites of the adenovirus shuttle plasmid pShuttle CMV-IRES-hrGFP—1by the directional cloning method.To identify the correct recombinants the analysis of sequencing and electrophoresis was adopted.Results The electrophoresis and sequencing map of the recombinant accorded with theoretic analyses of pShuttle CMV-~＋ VEGF_（121）-IRES-hrGFP-1.Conclusions The adenovirus shuttle plasmid pShuttle CMV-VEGF_（121）-IRES-hrGFP-1 has been constructed successfully.

关 键 词：腺病毒穿梭载体 血管内皮生长因子121 

分 类 号：R317.3[医药卫生—基础医学]