重组腺病毒载体Ad5-hVEGF165-EGFP的构建与鉴定

目的人血管内皮生长因子（的hVEGF165）为靶基因,增强型绿色荧光蛋白（EGFP）基因构建体的复制缺陷型腺病毒载体,为基因治疗牙周进一步再生奠定了基础。方法质粒的pDC316-的VEGF165为模板,PCR扩增基因片段进行消化,获得的hVEGF165用于连接到含有绿色荧光蛋白标记基因重组质粒的消化方法的pDC316-MCMV-EGFP质粒载体,PCR鉴定和双酶切证实成功构建重组质粒;使用ADMAX包装系统,改造的质粒共转染用质粒骨架293包装细胞系和重组病毒的扩增;扩增病毒的离子交换纯化;TCID50测定病毒粒子和效价的数目;的荧光的荧光显微镜重组腺病毒表达。结果 PCR鉴定,酶切分析和测序证实成功构建人VEGF基因的携带绿色荧光蛋白标记（的hVEGF165）重组质粒的pDC316-的hVEGF165-MCMV-EGFP和同源重组腺病毒Ad5的-的hVEGF165-EGFP的成功。扩增和纯化后,腺病毒颗粒的数量测量5.4×1011VP/mL,约2.0,为1.8×1010CCID50/mL的滴度OD260/OD280值。结论成功构建携带hVEGF165基因重组腺病毒载体Ad5-hVEGF165-EGFP并获得高滴度病毒颗粒,为hVEGF165基因功能研究以及细胞移植、基因治疗提供有效的工具。

Construction and Identification of Recombinant Adenovirus Vector Ad5-hVEGF165-EGFP

Objective This study is to made a recombinant adenovirus vector carrying human vascular endothelial growth factor 165（ hVEGF165） genes,linked with enhanced green fluorescent protein（ EGFP）,which can be used on the healing of periodontal tissue defects. Methods The hVEGF165 gene was constructed by PCR with plasmid pDC316-hVEGF165 as template. With enzyme digestion,the hVEGF165 gene was inserted into the vector pDC316-mCMV-EGFP and the recombinant plasmid pDC316-hVEGF165-mCMV-EGFP was constructed. The hVEGF165 gene of Ad-hVEGF165-EGFP vector was analyzed b plamid was determined by NotI and HindIII. pDC316-hVEGF165-CMV-EGFP plamid was co-transfected in 293 cells with the adenovirus skeleton plasmid pBHGlox_E1,3Cre to obtain defective recombinant adenovirus vector Ad-hVEGF165-EGFP,and then the recombinant adenovirus was propagated by infection of 293 cells repeatedly and purified by ion exchange method. The virus particles were counted and the titers were determined with TCID50. Results Both recombinant shuttle plasmid pDC316-hVEGF165-mCMV-EGFP and recombinant adenovirus vector Ad-hVEGF165-EGFP were successfully constructed and the gene of hVEGF165 was identified by PCR amplification,restriction enzyme analysis and gene sequencing. After propagation and purification,the virus particle count,OD260 / OD280 ratio and titer of recombinant adenovirus were 5. 4 × 1011VP /mL,2. 0 and 1. 8 ×1010CCID50 /mL respectively. Conclusions In this study the recombinant adenovirus vector Ad-hVEGF165-EGFP was successfully constructed.