小鼠IFN-γ真核表达载体的构建及其抗肿瘤效应研究

目的：构建小鼠IFN-γ的真核表达载体，将载体在体外转染小鼠腹腔MΦ，观察MΦ的抗肿瘤效应。在荷瘤小鼠腹腔内注射重组表达载体，观察抗肿瘤效应。 方法： RT-PCR扩增小鼠IFN-γ mRNA，将扩增的cDNA通过亚克隆重组到表达载体pcDNA3.1上。体外转染小鼠的腹腔MΦ，RT-PCR检测小鼠IFN-γ mRNA的表达，用转染的MΦ的培养上清培养另一组MΦ，MTT法检测MΦ对肿瘤细胞的杀伤活性。将重组质粒注射到荷瘤小鼠腹腔，观察小鼠腹水出现时间和存活时间。 结果: 将小鼠基因的开放阅读框（ORF）重组到真核表达载体pcDNA3.1内，测序证实和GenBank所公布序列相符。体外将IFN-γ基因导入MΦ内并表达，其培养上清对MΦ有激活作用，杀伤肿瘤细胞活性增强。腹腔内注射重组表达载体，荷瘤小鼠的腹水增长延迟、荷瘤生存时间延长。 结论： 构建的小鼠pcDNA3.1-IFN-γ表达载体体外转染腹腔MΦ并获表达，体内外实验显示有抗肿瘤细胞活性。

Construction of eukaryotic expression vector of mouse IFN- γ and its antitumor effect

AIM: To construct a mouse IFN-γ expression vector and observe the antitumor effects of mouse peritoneal macrophages transfected with IFN-γ in vivo and in vitro. METHODS: The IFN-γ mRNA was amplified by RT-PCR. The open reading frame of mouse IFN-γ gene was recombinanted with eukaryotic expression vector pcDNA3.1 through subcloning. Mouse peritoneal macrophages were transfected with recombinant vector pcDNA3.1-IFN-γ. The expression of INF-γ mRNA was measured by RT-PCR. Another group of peritoneal macrophages were cultured with the culture medium from pcDNA3.1-IFN-γ transfecting groups, and its antitumor effect was measured by MTT. pcDNA3.1-IFN-γ plasmid was peritoneally injected inte mouse with tumor. The appearance of ascites of pcDNA3.1-IFN-γ plasmid injected mice and survival time were observed. RESULTS: The mouse IFN-γ expression vector pcDNA3.1-IFN-γ was constructed. The sequence was demonstrated to be the same as on GenBank. The recombinant vector was introduced into mouse peritoneal macrophages. IFN-γ mRNA was detected by RT-PCR. The supernatant from cultured macrophages transfected with pcDNA3.1-IFN-γ plasmid stimulated the antitumor effects of the macrophages without transfection. The appearance of ascites in pcDNA3.1-IFN-γ plasmid injected mouse was delayed and survival time was longer than that in other groups. CONCLUSION: We have successfully constructed the mouse IFN-γ expression vector pcDNA3.1-IFN-γ. Mouse peritoneal macrophages transfected with pcDNA3.1-IFN-γ have antitumor effects in vivo and in vitro.