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目的:构建高靶向性基因转移及表达系统,并研究其介导HSV-tK/GCV自杀基因系统对肝癌细胞的体外杀伤作用。方法:通过重组技术分别构建anti-TfR ScFv-GAL4融合蛋白表达载体ScFv-GAL4-pET28a及合AFP启动子、GAL4特异性识别序列(GAL4rec)的HSV-tk真核表达载体pEBAF/tk-GAL4rec。IPTC诱导后,利用受体介导内吞作用介导pEBAF/tk-CAL4rec质粒转染表面均高表达TfR的人肿瘤细胞株HepG2,SMMC7721及A549。潮霉素筛选后,MTT法检测GCV对它们的杀伤作用。结果:双酶切鉴定、SDS-PAGE电泳及测序分别证明anti-TfR ScFv-GAL4融合蛋白及重组pEBAF/tk-CAL4rec真核表达质粒构建成功。体外杀伤实验中,高分泌AFP(845ng/ml)的HepG2/tk细胞对GCV很敏感,且抑制率与GCV浓度及作用时间呈正相关;而低分泌AFP(2ng/ml)的SMMC7721/tk细胞对GCV低度敏感;不分泌AFP的A549/tk细胞则不敏感。结论:双靶向组织特异性HSV-tk/GCV抗瘤系统构建成功,并显示出很好的靶向性。
OBJECTIVE: To construct a high-targeted gene transfer and expression system and to study its in vitro cytotoxicity against hepatocellular carcinoma cells mediated by HSV-tK / GCV suicide gene system. Methods: Recombinant HSV-tk eukaryotic expression vector pEBAF / tk-GAL4rec containing ScFv-GAL4-pET28a and AFP promoter and GAL4rec was constructed by recombinant technique. After induced by IPTC, the human tumor cell lines HepG2, SMMC7721 and A549 with highly expressed TfR on the surface were transfected with plasmid pEBAF / tk-CAL4rec mediated by receptor-mediated endocytosis. Hygromycin screening, MTT assay GCV killing effect on them. Results: Double enzyme digestion, SDS-PAGE electrophoresis and sequencing proved that the anti-TfR ScFv-GAL4 fusion protein and the recombinant pEBAF / tk-CAL4rec eukaryotic expression plasmid were successfully constructed. In vitro cytotoxicity assay, HepG2 / tk cells secreting AFP (845ng / ml) were sensitive to GCV and the inhibitory rate was positively correlated with GCV concentration and time. SMMC7721 / tk cells secreting AFP (2ng / ml) GCV is low sensitive; A549 / tk cells that do not secrete AFP are not sensitive. Conclusion: The double-targeting tissue-specific HSV-tk / GCV anti-tumor system was successfully constructed and showed good targeting.