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构建由肝癌细胞专一的ａｆｐ基因表达调节元件控制自杀基因ＨＳＶ－ｔｋ的穿梭质粒，将它与缺陷型腺病毒载体重组，得到ＡｄｒＡＦＰＴＫ病毒。经ＰＣＲ及Ｓｏｕｔｈｅｒｎ杂交等证实它们含ａｆｐ元件和ｔｋ基因。空斑形成试验表明病毒效价达１×１０１５ｐｆｕ／Ｌ。同时构建由ＣＭＶ启动子控制ｔｋ基因的类似载体作为对照。将这两个重组腺病毒分别感染ＡＦＰ阳性（ＨｅｐＧ２）或阴性（ＨｅＬａ，ＢＲＬ－３Ａ）细胞株（ｍ．ｏ．ｉ．＝１００），以丙氧鸟苷（ｇａｎｃｉｃｌｏｖｉｒ，ＧＣＶ）处理后，用ＭＴＴ法测定杀伤细胞的效应。结果，ＡｄＣＭＶＴＫ感染这三种细胞后，ＧＣＶ半杀伤浓度分别为１．３、２、＜１μｍｏｌ／Ｌ；但是，ＡｄｒＡＦＰＴＫ感染的ＨｅＬａ和ＢＲＬ－３Ａ细胞的ＧＣＶ半杀伤浓度都＞１０００μｍｏｌ／Ｌ，而对ＨｅｐＧ２细胞只有＜１μｍｏｌ／Ｌ，表现出极高的细胞专一性。重组腺病毒ＡｄｒＡＦＰＴＫ可望用于肝癌的专一性基因治疗
A shuttle plasmid targeting the suicide gene HSV-tk was constructed by a hepatoma cell-specific afp gene expression regulatory element, which was recombined with the defective adenovirus vector to obtain the AdrAFPTK virus. They were confirmed by PCR and Southern hybridization to contain afp element and tk gene. Plaque formation assays demonstrated viral titer of 1 x 1015 pfu/L. At the same time, a similar vector that controls the tk gene by the CMV promoter was constructed as a control. The two recombinant adenoviruses were infected with AFP-positive (HepG2) or negative (HeLa, BRL-3A) cell lines (m.o.i. = 100) and treated with ganciclovir (GCV). The effect of killer cells was determined by MTT assay. As a result, the GCV quasi-lethal concentrations of AdCMVTK were 1.3, 2, and <1 μmol/L, respectively, after infection with these three cells; however, GCV halal concentrations of AdrAFPTK-infected HeLa and BRL-3A cells were >1000 μmol/L. Only <1 μmol/L for HepG2 cells, showing very high cell specificity. Recombinant adenovirus AdrAFPTK is expected to be used for specific gene therapy of liver cancer