携带人白细胞介素10和肝细胞生长因子双基因的重组腺病毒载体构建与鉴定

Construction and identification of recombinant adenovirus expression vector of hIL-10 and hHGF double genes

目的:构建携带人白细胞介素10(hIL-10)和人肝细胞生长因子(hHGF)双基因的重组腺病毒载体。方法:分别以pDC316-hIL10-EGFP和pCDNA3-hHGF重组质粒为模板,PCR扩增获得hIL-10和hHGF基因片段,与pIRES连接成为hIL10-IRES-hHGF,测序正确后酶切,与pDC315连接,获得穿梭质粒pDC315-hIL10-IRES-hHGF,与腺病毒包装系统转染293细胞,包装产生重组腺病毒hIL10-hHGF,PCR鉴定后,扩增病毒,并测定病毒滴度。用重组腺病毒转染BEL-7402和WRL-68细胞,ELISA法检测上清液中hIL-10和hHGF的含量。结果:成功构建了重组腺病毒hIL10-hHGF,扩增纯化后测得重组腺病毒滴度为1×109pfu/ml,转染BEL-7402和WRL-68细胞72 h后,表达hIL-10的量分别为(6 271.7±5.2)pg/ml和(350.6±12.4)pg/ml,表达hHGF的量分别为(20 827.1±345.5)pg/ml和(201.6±10.1)pg/ml。结论:本实验为进一步研究Ad-hIL10-hHGF在动物体内抗肝炎、肝纤维化作用奠定了基础。

Objective： To construct the recombinant adenovirus vector carrying human interlerkin-10（hIL- 10） and human hepatocyte growth faetor（hHGF）. Methods： We amplified hIL-10 and hHGF genes by PCR with plasmids pDC316-hIL10-EGFP and pCDNA3-hHGF, respectively, and subeloned into pIRES to generate hIL10-IRES-hHGF. Confirmed by sequence analysis, the hIL-10-IRES-hHGF genes were inserted into the vector pDC315, then the shuttle plasmid pDC315-hIL10-IRES-hHGF was constructed, which was cotransfected with the adenovirus skeleton plasmid into 293 ceils to obtain the recombinant adenovirus vector hIL10-hHGF. After identified the target genes by PCR, the recombinant adenovirus was propagated and the viral titer was determined, hIL-10 and hHGF proteins in the virus-infected BEL-7402 and WRL-68 cells were measured by ELISA. Results： We obtained recombinant adenovirus vector of hIL10-hHGF successfully. After propagation, the titer of the recombinant adenovirus was 1 ×10^9 pfu/ml. The recombinant adenovirus could express hIL-10 and hHGF in BEL-7402 and WRL-68 cells effectively. The concentrations of hIL-10 were（6 271.7± 5.2 ） pg/ml and （350.6 ± 12.4） pg/ml respectively, and the concentrations of hHGF were （20 827.1 ±345.5） pg/ml and （201.6 ±10. 1 ） pg/ml respectively. Conclusion： The recombinant adenovirus vector hIL10-hHGF seted the foundation for the gene therapy of hepatitis and cirrhosis in animals.