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  

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

作　　者：徐震[1] 牛坚[2] 刘斌[2] 

机构地区：[1]徐州医学院研究生院,江苏徐州221002 [2]徐州医学院附属医院普外科,江苏徐州221006

出　　处：《中国医药科学》2011年第7期17-21,共5页China Medicine And Pharmacy

基　　金：江苏省高校自然科学研究项目资助(编号09KJB320018)

摘　　要：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。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.

关 键 词：RNA干扰 单链抗体 人胰岛素样生长因子1类受体 慢病毒 

分 类 号：R394[医药卫生—医学遗传学]