人AFP启动子操控的跨膜型抗CD3单链抗体构建及其在AFP阳性肝癌细胞中特异性表达

Preparation of transmembrane anti-CD3 single chain Fv antibody drived by human AFP promoter and its specific expression in AFP positive liver cancer cells

目的设计肝癌特异性人甲胎蛋白启动子(hAFPp)操控的跨膜型抗CD3单链抗体(antiCD3scfv),使其仅在AFP阳性(AFP+)肝癌细胞中特异性表达。方法利用分子克隆技术构建pAd-hAFPp-CD3scfv腺病毒载体、pAd-hAFPp-antiCD3scfv穿梭质粒载体,经测序验证其基因序列的正确性。取AFP+人肝癌细胞HepG2、Huh-7及AFP阴性(AFP-)正常人肝细胞HL-7702、人乳腺癌细胞MCF-7,利用双荧光素酶报告基因检测系统鉴定hAFPp的肝癌细胞转录特异性;通过Western blotting、免疫荧光、流式分析检测antiCD3scfv蛋白在细胞的表达、定位及表达效率。结果构建的pAd-hAFPp-CD3scfv腺病毒载体、pAd-hAFPp-antiCD3scfv穿梭质粒载体,经测序后基因序列正确。hAFPp在AFP+的HepG2、Huh-7细胞中转录活性分别为160.86%±26.24%、80.08%±12.64%,而在AFP-细胞中转录活性均<7%(P均<0.05)。在AdCD3scfv感染的细胞中,仅HepG2、Huh-7细胞中检测到antiCD3scfv蛋白表达(36.7 kD),且表达于细胞膜表面,细胞中GFP+antiCD3scfv+细胞群比例>90%,而HL-7702、MCF-7均无条带出现。结论携带antiCD3scfv蛋白基因的腺病毒感染周围细胞后,hAFPp调控antiCD3scfv在肝癌细胞的细胞膜特异性表达,进而可能实现antiCD3scfv分子直接介导免疫杀伤的抗肿瘤效果。

Objective To design a transmembrane anti-CD3 single chain Fv antibody(antiCD3scfv)drived by a liver-specific human alpha-fetoprotein promoter(hAFPp),so that it can only be expressed on AFP positive liver cancer cells.Methods Molecular cloning technique was used to construct the adenoviral vector pAd-hAFPp-CD3scfv and plasmid shuttle vector pAd-hAFPp-antiCD3scfv,and the gene sequence was verified by sequencing.AFP positive hepatoma cells HepG2 and Huh-7,AFP negative human normal liver cells HL-7702 and human mammary carcinoma cells MCF-7 were used to verify the specific activity of hAFPp promoter by dual luciferase assay.Western blotting,flow cytometry,and immunofluorescence analysis were performed to detect the expression,localization and expression efficiency of antiCD3scfv protein in cells.Results The adenoviral vector pAd-hAFPp-CD3scfv and plasmid shuttle vector pAd-hAFPp-antiCD3scfv were successfully constructed,and the gene sequence was correct.The transcriptional activities of hAFPp in HepG2 and Huh-7 cells were 160.86%±26.24%and 80.08%±12.64%,while the transcriptional activities in AFP negative cells were<7%(all P<0.05).In AdCD3scfv-infected cells,antiCD3scfv protein expression was only detected in HepG2 and Huh-7 cells(36.7 kD)and expressed on the cell membrane surface.The proportion of GFP+antiCD3scfv+population in cells was greater than 90%,while no protein product was observed in HL-7702 and MCF-7 cells.Conclusion After adenovirus carrying antiCD3scfv protein genes infect the cells around,hAFP promoter regulates membrane specific expression of hepatoma cells;moreover it is possible to achieve the anti-tumor effect of antiCD3scfv molecules directly mediating immune killing.