人IFN-γ cDNA逆转录病毒载体的构建及其在人膀肤癌细胞中的表达

Expression of Human IFN-γ in Human Bladder Carcinoma Cells after Retroviral Vector Mediated Gene Transfer

用逆转录病毒载体介导转移人干扰素γ(hIFN-γ)基因在人膀胱癌细胞EJ中稳定表达。应用基因重组技术构建含hIFN-γ基因的逆转录病毒载体pZip-hIFN-γ,采用脂质体介导法转染包装细胞Ψ-2和PA 317,然后用高病毒滴度的PA 317细胞培养上清感染EJ细胞。经G 418筛选培养得到一株能稳定分泌hIFN-γ(210IU/ml)的EJ克隆,Southern印迹证实hIFN-γ基因已插入EJ基因组中。逆转录病毒载体pZip-hIFN-γ能有效地介导基因转移,并能使目的基因在靶细胞稳定表达,这为人膀胱癌转hIFN-γ基因瘤苗的应用研究打下了基础。

The aim is to express human IFN-Y in EJ cells of human bladder carcinoma by retroviral vector-mediated gene transfer. Retroviral vector containing human IFN-γ cDNA (named pZip-IFN-γ)was con structed using gene recombinant techniques. Packaging cell lines Ψ-2 and PA 317 were transducted with pZip-hIFN-γ. The supernatant of PA 317 cells containing high titier of virus were used to infect EJ cells. On G418-Selecting culture, G418-resistant clonies were obtained and hIFN-γ was highly expressed up to 210 In ml as detected by IFN-γ activity assay. Southern blot hybridization demostrated that human IFN-γ gene was integrated in the genome of EJ Ccells. Retroviral vector pZip-hIFN-γ could effectively mediat gene transfer to human cells and promote stable expression of therapeatic gene in the target cells. This laid the foundation to develop hIFN-γ gene-transducted tumor vaccine of human bladder cancer.