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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 highly biologically safe eukaryotic expression vector for human endostatin and to lay the foundation for the anti-angiogenic agent to transfect tumor cells. Methods: Endostatin gene with human IgGγ chain signal sequence, KpnI, EcoRI double digestion vector and PCR product were amplified by PCR using the eukaryotic expression plasmid pVAX1 which was approved by human beings. The PCR product was ligated with T4 DNA ligase, Construction of recombinant expression vector, named pVAX Juan sEN. The recombinants were identified by KpnI, EcoRI digestion, PCR and sequencing. The correct recombinant vector was transfected into human hepatocellular carcinoma cell line HepG2 by liposome, and the expression of Endostatin was identified by RT-PCR and ELISA. Results: 610bp Endostatin gene with signal peptide was obtained by PCR. Sequencing results showed that eukaryotic expression vector pVAX-sEN containing signal peptide and Endostatin was correctly constructed. After transfected HepG2 cells with recombinant plasmid for 72h, RT-PCR showed Endostatin mRNA expression. ELISA confirmed that HepG2 cells transfected with the recombinant vector highly expressed protein of interest, the expression amount of 172.34ng / ml. Conclusion: The recombinant plasmid pVAX-sEN was successfully constructed, which laid the foundation for the gene therapy research of solid tumors such as liver cancer.