高生物安全性内皮抑素真核表达载体的构建

Construction of recombinant plasmid with human endostatin cDNA in high-biosafety

目的:构建具有高度生物安全性的人内皮抑素(Endostatin)真核表达载体,为抗血管生成剂转基因治疗肿瘤奠定实验基础。方法:采用被FDA批准的可用于人体实验的真核表达质粒pVAX1为载体,PCR扩增带有人IgGγ链信号肽序列的Endostatin基因,KpnI、EcoRI双酶切载体和PCR产物,T4DNA连接酶连接,构建重组表达载体,命名为pVAX鄄sEN。重组子经KpnI、EcoRI双酶切、PCR及测序鉴定。将测序正确的重组载体经脂质体转染体外培养的人肝癌细胞系(HepG2),用RT鄄PCR、ELISA方法鉴定Endostatin的表达。结果:PCR获得了610bp带有信号肽的Endostatin基因,测序结果表明,正确构建了含有信号肽及Endostatin的真核表达载体pVAX鄄sEN。重组子转染HepG2细胞72h后,RT鄄PCR显示有EndostatinmRNA表达。ELISA证实,转染重组载体的HepG2细胞上清有目的蛋白的高效表达,表达量为172.34ng/ml。结论:成功构建了高生物安全性真核表达载体pVAX鄄sEN,为肝癌等实体瘤的基因治疗研究奠定了基础。

Objective: To construct a high-biosafety eukaryotic expression vector carrying human endostatin cDNA and exploit the potential of gene therapy. Methods: pVAX1, which was authorized by FDA to be used in clinical trial, was taken as the expression vector. Endostatin gene with signal sequence of human IgGγ chain was amplified by PCR. The PCR products digested by EcoRI and KpnI were cloned into pVAX1 to construct a recombinant plasmid, named as pVAX-sEN. The recombinant plasmid was detected with EcoRI/KpnI, PCR and DNA sequencing. Then pVAX-sEN was transfected into HepG2 cells by Lipofectamine 2000. The expression of Endostatin in HepG2 was detected by RT-PCR and ELISA. Results: Endostatin gene together with signal sequence was amplified by PCR. EcoRI/KpnI, PCR and DNA sequencing indicated the recombinant plasmid was correctly cloned. The recombinant plasmid was transfected into HepG2 cells. Seventy-two hours after the transfection the HepG2 cells and cell culture medium were collected. RT-PCR showed the Endostatin mRNA was expressed. The highest concentration of endostatin in culture medium was 172.34ng/ml. Conclusion: The high-biosafety recombinant plasmid pVAX-sEN was successfully constructed which lays a foundation for further research on gene therapy for tumor using anti-angiogenesis agents.