携带人肝细胞生长因子基因重组腺病毒的构建与制备

构建一种携带人肝细胞生长因子基因的重组腺病毒(AdHGF),并对其进行扩增、纯化与质量检测。首先构建携带人肝细胞生长因子基因的穿梭质粒pXCJL1CMV/pAHGF,然后用LipofectAMIN介导该质粒和含有E1、E3区及包装信号区缺失的复制缺陷型5型腺病毒基因组的质粒GT4050共转染293细胞,采用细胞内质粒DNA同源重组法构建重组腺病毒(AdHGF)。并以噬斑分析法筛选单克隆重组腺病毒,PCR法鉴定阳性重组腺病毒,氯化铯密度梯度离心法纯化病毒,紫外分光光度仪测定病毒颗粒数及纯度,快速CPE分析、噬斑分析法测定病毒感染滴度(pfu/ml)。采用敏感细胞病变法检测有复制能力的腺病毒。结果成功构建了重组腺病毒AdHGF,制备的病毒纯度好、滴度高,其效价比小于1∶100,并且未检测到有复制能力的腺病毒存在。研究构建制备的重组腺病毒AdHGF具有潜在的实际应用价值。

Preparation and Construction of a Recombinant Adenovirus Carrying Human Hepatocyte Growth Factor Gene

The purpose is to construct a recombinant adenovirus carrying human hepatocyte growth factor(HGF) gene (Ad-HGF). Then it was multipiled and purified and its quality was evaluated. To construct a shuttle recombinant plasmid carrying HGF gene (pXCJL1-CMV/HGF/pA), the plasmid was subjected to co-transfection with another plasmid GT4050 containing replication-defective Ad5 lacking E1 and E3~( )domains in 293 cells. This recombinant replication-defective adenovirus (Ad-HGF) was recovered by homogenous recombination. The presence of HGF cDNA in the viral genome was verified by PCR. Ad-HGF was multiplied to a large quantity in 293 cells growing in DMEM containing 5% FBS. Then Ad-HGF was purified by cesium chloride gradient centrifugation and desalted using Dialysis Cassettes. The particle number and the purity of Ad-HGF were determined by absorption at A_(260()nm) and the ratio of A_(260()nm)/A_(280()nm) using a DU-640 spectrophotometer. The final plaque-forming units (PFU) were determined by titration on 293 cells under an agar overlay (Low Gelling Temperature, Sigma). The presence of replication-competent adenovirus was detected. A recombinant replication-defective adenovirus carrying HGF gene was successfully constructed. The adenovirus has good purity and high viral titer, and the replication-competent adenovirus was not detected. The Ad-HGF constructed and prepared here has a potential application value.