腺病毒穿梭质粒pshuttle cmv- vegf121-ires-hrgfp-1的构建和鉴定.docVIP

腺病毒穿梭质粒pShuttle CMV- +VEGF121-IRES-hrGFP-1的构建和鉴定 【摘要】 目的 构建腺病毒穿梭质粒pShuttle CMV-+VEGF121-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,VEGF121)，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-VEGF121携带的VEGF121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-VEGF121携带的VEGF121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VEGF121基因(+VEGF121)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- +VEGF121-IRES-hrGFP-1。 【关键词】 腺病毒穿梭载体;血管内皮生长因子121 Abstract: Objective To clone vascular endothelial growth factor 121 (VEGF121) gene into the adenovirus shuttle plasmid pShuttleCMV-IRES-hrGFP-1, preparing for construction of a novel recombinant adenovirus vector expressing the VEGF121 fused to FLAG epitope and humanized Renilla reniformis green fluorescent protein 1(hrGFP-1) as a reporter on the same transcript. Methods The VEGF121 gene contained in the plasimid of pTG19T-VEGF121 was sequenced, and the profile of restriction endonuclease sits existing in the sequence was analysed. Then the translation stop codon TAG was removed. Meanwhile, NotI 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 VEGF121 gene(+VEGF121 gene) was ligated into the multiple cloning sites of the adenovirus shuttle plasmid pShuttle CMV-IRES-hrGFP一1by 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-+ VEGF121-IRES-hrGFP-1. Conclusions The adenovirus shuttle plasmid pShuttle CMV- VEGF121-IRES-hrGFP-1 has been constructed successfully. 　　Key words:adenovirus shuttle plasmid; vascular endothelial growth factor 121(VEGF121) 　　在骨组织的胚胎发生或骨损伤愈合的过程中，缺乏良好的血供将