肝癌特异性启动子调控的双靶位慢病毒载体的构建、鉴定及其在肝癌基因治疗中的应用

目的探讨survivin-CMV特异性启动子调控的双靶点慢病毒对肝癌细胞生长的影响。方法 RT-PCR扩增anti-AFP-scFv,测序鉴定,双酶切连接,获得pMD2G-anti-AFP-scFv质粒。针对已经筛选确定的IGF1R基因干扰有效序列,合成靶序列的OligoDNA,退火形成双链DNA,与质粒pPRIME连接得到pPRIME-miR30-shRNA-IGF1R质粒。RT-PCR扩增survivin-CMV肝癌特异性启动子,测序鉴定,与双酶切的质粒pPRIME-miR30-shRNA-IGF1R连接,得到质粒sur-CMV-pPRIME-miR30-shRNA-IGF1R。用sur-CMV-pPRIME-miR30-shRNA-IGF1R, psPAX2, pMD2G-anti-AFP-scFv共转染293T细胞,经过病毒空斑纯化,包装成慢病毒,测定病毒滴度。RT-PCR、Westernblot检测IGF1R表达,竞争抑制实验检测抗人AFP单链抗体活性。CCK-8法检测细胞生长。结果成功构建survivin-CMV肝癌特异性启动子调控的慢病毒AFP-sur-CMV-PRIME-miR30-shRNA-IGF1R;滴度为4.58×109PFU/mL;AFP-sur-CMV-PRIME-miR30-shRNA-IGF1R抑制了肝癌细胞IGF1R表达,竞争实验显示抗人AFP单链抗体的基因高效表达,该慢病毒抑制肝癌细胞的生长。结论本次实验构建的慢病毒载体具有良好的靶向性,为肝癌生物治疗奠定了理论基础,提供了新的思路。

Construction and Identification of Lentiviral Vector of Double Targeting Regulated by Special Promoter of Liver Cancer and Application of Gene Therapy of Liver Cancer

Objective To study the interferencing and anti-tumor effects of lentiviral vector of double Targeting liver Cancer Cell regulated by special survivin-CMV promoter. Methods The fragment of the anti-AFP-scFv was acquired by PCR to recombinant plasmid pMD2G-anti-AFP-scFv. The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed, synthesized and cloned into the pPRIME vector,named pPRIME-miR30-shRNA-IGF1R. The fragment of the special survivin promoter was acquired by PCR amplification and inserted into pPRIME-miR30-shRNA-IGF1R,to recombinant plasmid sur-CMV-pPRIME-miR30-shRNA-IGF1. The effect of AFP-PRIME-miR30-shRNA- IGF1R on IGF1R expression of SMMC7721 cells was detected by RT-PCR and Westernblot. Competitive-inhibitive expermient checks the activity of aiti-AFP-scFv. The antitumor potential of AFP-PRIME-miR30-shRNA-IGF1R to SMMC7721 cells was evaluated by CCK-8 assay. Results AFP-sur-CMV-PRIME-miR30-shR-NA-IGF1R were constructed successfully. Functional PFU titers of AFP-sur-CMV-PRIME-miR30-shRNA-IGF1R were 4.58×109PFU/mL.AFP-sur-CMV-PRIME-miR30-shRNA-IGF1R was more effective to inhibit IGF1R expression in mRNA or protein levels and the proliferation of SMMC7721 cells. The experiment showed the higher effect of aiti-AFP-scFv gee. It can inhibit IGF1R expression. Conclusion The lentiviral vector of construction holds better Targeting,it may be used for further investigation of Gene therapy of liver cancer.