Construction of pEGFP-Nl-linamarase fusion gene in eukaryotic expression vector and its expression in HepG2 Cells

Objective To construct pEGFP-Nl-linamarase eukaryotic expression vector by using green fluo-rescence protein as reporter gene to establish the linamarase/linamarin tumor-killing system of gene therapy and investigate its killing effect on MHCC97 cells in vitro. Methods The cassava tissue linamarase （Lis） genes were amplified with PCR technique and inserted into pEGFP-N1 vector. The HepG2 ceils were transfected with the formed plasmid by means of electrotransfer. The linamarase-EGFP fused protein was viewed directly with fluorensce microscope, and the expression of linamarase was detected by RT-PCR （reverse transcription-PCR） and western-blot. Results The plasmid was formed correctly and detected hy PCR. Meanwhile, it was sequenced and expressed in the HepG2. The fused protein had activities of both linamarase and EGFP. Conclusion HepG2 cells modified bv Lis gene can play a role in the treatment of tumor injury. Key words: Carcinoma, hepatocellular;  Linamarase/Linamarin, lis/lin;  PCR;  Gene transfer technique