肝细胞肝癌靶向性r-Caspase-3重组腺病毒的构建与表达

Construction and Expression of The Recombinant of Hepatocellular Carcinoma-Targeting Adenovirus Containing r-Caspase-3 Gene

目的构建肝细胞肝癌特异性表达反向半胱氨酸蛋白酶3(r-Caspase-3)重组腺病毒,为肝细胞肝癌的基因治疗提供新策略。方法构建甲胎蛋白(AFP)增强子和白蛋白(ALB)启动子腺病毒载体(pAdTrack-EAFP-PALB),然后将目的基因r-Caspase-3亚克隆到载体pAdTrack-EAFP-PALB上,获得重组腺病毒穿梭载体pAdTrack-EAFP-PALB/r-Caspase-3,经PmeⅠ酶切线性化后与pAdEasy-1同源重组,获得重组腺病毒骨架pAdEasy-EAFP-PALB/r-Caspase-3;鉴定正确的pAdEasy-EAFP-PALB/r-Caspase-3经PacⅠ酶切线性化后脂质体转染AD293细胞进行包装、扩增,获得病毒。绿色荧光蛋白(green fluorescent protein,GFP)监测病毒滴度和感染效率;RT-PCR和West-ernblot法检测r-Caspase-3在HepG2细胞中的表达;SRB染色法评估重组腺病毒对HepG2细胞的抑制作用,初步观察HepG2细胞凋亡状况。结果穿梭载体pAdTrack-EAFP-PALB/r-Caspase-3酶切、测序正确。穿梭载体、pAdEasy-1载体同源重组后PCR及PacⅠ酶切鉴定结果表明pAdEasy-EAFP-PALB/r-Caspase-3重组成功;经PacⅠ酶切线性化后,pAdEasy-EAFP-PALB/r-Caspase-3转染AD293细胞即可观察到GFP的表达;回收病毒可重复感染AD293细胞,RT-PCR和Western blot均可检测到r-Caspase-3的表达,证实Ad-EAFP-PALB/r-Caspase-3病毒颗粒包装成功;SRB染色检测发现Ad-EAFP-PALB/r-Caspase-3具有凋亡诱导特异性。结论靶向性Ad-EAFP-PALB/r-Caspase-3重组腺病毒构建成功,并具有凋亡诱导靶向性,为进一步研究靶向性r-Caspase-3基因治疗肝细胞肝癌及其生物学功能奠定了基础。

Objective To construct the recombinant of hepatocellular carcinoma-targeting adenovirus contai-ning r-Caspase-3 gene and provide the gene therapic strategy for hepatocellular carcinoma. Methods The pAdTrack-EAFv-PALB was constructed and the r-Caspase-3 gene was subcloned into the vector. The linearized shuttle plasmid was homogenously recombined with AdEasy-1 in BJ5183 cells. The candidate clone was analyzed by restriction endonuclease digestion and sequencing, and then pAdEasy-EAFP-PALB/r-Caspase-3 vector was digested with Pac I and transfected into AD293 cells for packaging and amplifying, recombinant virus was constructed successfully. Infection titer and efficiency of recombinant virus were monitored by green fluorescent protein （GFP） expression. The expression of r-Caspase-3 in infected HepG2 cells was detected by RT-PCR and Western blot. The apoptosis of HepG2 cells was detected by SRB dyeing method. Results Shuttle vector pAdTrack-EAFP-PALB/r-Caspase-3 was correct after identification by restriction endonuclease analysis and sequencing. By PCR and Pac I restriction endo-nuclease analysis, the homologous recombinant of pAdEasy-EAFP-PALB/r-Caspase-3 was successful. The expression of GFP was observed when linearized pAdEasy-EAFP-PALB/r-Caspase-3 was transfected into AD293 cells. AD293 cells could be infected repeatedly by recombinant adenovirus. The expression of r-Caspase-3 gene on HepG2 cells was detected by RT-PCR and Western-blot methods respectively, which confirmed that the Ad-EAFP-PALB/r-Caspase-3 was constructed successfully. The specificity of Ad-EAFP-PALB/r-caspase-3 which targeting induced hepatocellular carcinoma cells was founded by SRB dyeing test. Conclusion The Recombinant of hepatocellular carcinoma-targeting adenovirus containing r-Caspase-3 gene was constructed successfully and which established the foundation of r-Caspase-3 gene therapy in future research to hepatocellular carcinoma.