肝癌组织特异性P_(afp)IRES2-EGFP表达载体的构建及表达

Construction of Murine Hepatoma Specific P_(afp)IRES2-EGFP Vector and Its Expression in H22 Cells

目的构建AFP最小启动子调控的小鼠肝癌组织特异性表达载体,观察其在小鼠H22肝癌细胞中的特异性表达。方法从H22细胞总DNA和荧光载体pIRES2-EGFP中分别PCR扩增AFP最小启动子区序列和CMV增强子(ECMV),利用重叠延伸PCR方法(SOE)获得AFP启动子驱动的嵌合调控序列,将其克隆入pIRES2-EGFP载体,进行菌落PCR、限制性酶谱分析、DNA序列测定。通过jetPEI方法转染H22肝癌细胞和YAC1淋巴瘤细胞,荧光显微镜下观察其表达的组织特异性。结果从小鼠H22细胞DNA和pIRES2-EGFP中分别扩增得到229bp和324bp的片段,纯化后的PCR产物通过SOE扩增得到一537bp的条带,均与预期序列长度一致。SOE产物与pIRES2-EGFP连接,菌落PCR鉴定获得数个阳性克隆,质粒经Nde I+NheⅠ限制性酶谱分析酶解出537bp条带,DNA序列分析证实重组载体中的嵌合DNA与GeneBank中小鼠AFP启动子和ECMV序列完全一致。结论本研究成功构建了AFP启动子调控的小鼠肝癌组织特异性表达载体PafpIRES2-EGFP,该载体能够在小鼠肝癌细胞中特异性表达外源基因。

Objective To establish a novel minimal AFP promoter operated hepatoma specific expression vector and analysis its specific expression in H22 cells. Methods Genomic DNA from H22 hepatoma cells was used to amplify the minimal AFP promoter through PCR, CMV enhancer was prepared by PCR using pIRES2-EGFP as template. The two DNA fragments were spliced through overlapping PCRto form a chimeric promoting sequence. Purified product digested by Nde I and Nhe I was cloned into pIRES2-EGFP to construct the minimal AFP promoter operated murine hepatoma specific PafpIRES2-EGFP expression vector which was verified by PCR screening, restriction enzyme assay（ Nde I and Nhe I ） and DNA sequencing. Then purified Pafp IRES2-EGFP plasmid was transfected into H22 hepatoma cells and YAC- 1 lymphoma cells through a jetPEl mediated method. Tissue specific expression the recombinant vector was analyzed by fluorescent microscopy. Results A 229bp and a 324bp DNA segments were amplified respectively from genomic DNA of H22 cells and pIRES2-EGFP. Purified PCR products when spliced by SOE PCR which produced a chimeric DNA sequence of 537bp in length as expected. SOE product and pIRES2-EGFP digested with Nde I and Nhe I respectively were ligated together by Tn liagase,in the following colony PCR it was showed that we got several positive clones,when further assessed by restriction enzyme assay, they gave rise to a 537bp DNA fragment which was finally proved to be identical to the minimal AFP promoter and ECMV. The recombinant vector were then transfected into H22 hepatoma cells and YAC-1 cells by jetPEI,bright green fluorescence could be observed only on the cell surface of H22 cell rather than YAC-I cells at 48h after transfection. Conclusion The Authors successfully constructed a murine hepatoma specific expression vector Pafp IRES2-EGFP operated by the minimal AFP promoter,the recombinant vector could express exogenous gene effectively in murine hepatoma cells.