目的 构建表达亚麻苦甙水解酶(linamarase,lis)基因的重组真核载体,建立亚麻苦甙水解酶/亚麻苦甙(linamarase/linamarin,lis/lin)肿瘤自杀基因治疗系统并检测其对肝癌细胞MHCC97的体外杀伤效应.方法 将lis基因定向克隆入真核载体pEGFP-N1,并用限制性内切酶进行酶切及测序鉴定;用电转染法将pEGFP-Nl-lis转染入肝癌细胞MHCC97,通过G418筛选建立稳定表达lis的细胞系,MTT法检测lis/lin自杀基因治疗系统在体外的抗肿瘤效应.结果 经酶切鉴定和DNA序列测定,证明lis基因成功定向插入到pEGFP-Nl载体中;G418压力筛选4周,得到稳定表达lis的MHCC97细胞,命名为MHCC97-lis.经检测,新型自杀基因系统lis/lin对肿瘤细胞有较强的杀伤作用并显示出旁观者效应.结论 成功获取表达lis基因的真核表达载体,已建立显示出对肿瘤细胞的杀伤作用以及旁观者效应的自杀基因治疗系统,为进一步研究新型自杀基因lis/lin抗肿瘤作用奠定基础.%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 tis-sue linamarase (Lis) genes were amplified with PCR technique and inserted into pEGFP-N1 vector. The HepG2 cells were transfected with the formed plasmid by means of electrotransfer. The linama-rase-EGFP fused protein was viewed directly with fluorensce microscope, and the expression of linamarase was detected by RT-PCR (reverse transeription-PCR) and western-blot. Results The plasmid was formed correctly and detected by PCR. Meanwhile, it was sequenced and expressed in the HepG2. The fused protein had activities of both linamarase and EGFP. Conclusion HepG2 cells modified by Lis gene can play a role in the treatment of tumor injury.
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