小鼠IL-18 cDNA克隆及在H_(22)肝癌细胞中的初步表达  

Murine interleukin-18 cDNA cloning and its expression in murine H22 hepatocarcinoma cell

作　　者：周 虎[1] 陆 梁[1] 裴冬生[1] 赵惠仁[1] 

机构地区：[1]徐州医学院生物化学与分子生物学研究中心,江苏徐州221002

出　　处：《免疫学杂志》2002年第3期221-224,共4页Immunological Journal

摘　　要：目的 构建小鼠白细胞介素-18（mIL-18）逆转录病毒载体,实现mIL-18在小鼠H22肝癌细胞中表达。方法 用RT-PCR方法从小鼠肝脏细胞中扩增出mIL-18 cDNA,克隆到载体pBluscript中,测序后亚克隆到逆转录病毒载体pLNCX中。pLNCX/mIL-18重组子用脂质体介导的方法转染PA317包装细胞,用400μmL G418进行筛选。挑取筛选得到的阳性克隆扩大培养,取上清感染NIH3T3细胞和H22肝癌细胞。用感染后的H22细胞上清诱导小鼠脾细胞IFN-r生成能力来检测H22细胞分泌mIL-18的情况。结果 用NH3T3细胞测得病毒滴度为1.5×105克隆形成单位CFU/mL,转染后的H22肝癌细胞上清对小鼠脾细胞具有显著的IFN-r诱生作用。结论 pLNCX/mIL-18重组子构建成功,初步证明转染后的H22肝癌细胞能够表达IL-18。ve To study the expression of murine interleukin-18 (mIL-18) in eucaryotic cells transfected with retroviral vector encoding mIL-18 cDNA, which is the first step towards mIL-18 gene therapy of cancer. Methods Total RNA was extracted from murine hepatic cells and cDNA of mIL-18 was amplified by RT-PCR. The cDNA was introduced into the vector pBluscript and then subcloned into the retroviral vector pLNCX. The pLNCX/mIL-18 vector was transduced into PA317 cells by cationic liposomes and packaged. After selected in medium sup-plemented with 400μg/mL G418, the G418-resistant colonies were isolated and expanded. NIH3T3 and H22 cells were transfected with superna-tant containing pseudotyping retrovirus and underwent selection with G418. The activity of mIL-18 secreted from transfected H22 cells was deter-mined by an enzyme-linked immunosorbent assay (ELISA) of IFN-r production induced by mIL-18. Results The titer of the retroviral superna-tant was 1.5 × 105 CFU/mL. In the presence of 0.15 mg/L Con A, the supernatant collected from conditioned media of the H22 cells transfected with mIL-18 encoding retrovirus enhanced IFN-r production from murine splenocytes. Conclusion We have constructed the recombinant pLNCX/mIL-18 plasmid and the transfected H22 cells expressed mIL-18 in vitro.

关 键 词：白细胞介素-18 逆转录病毒载体 克隆 CDNA 肝癌 癌细胞 动物实验 

分 类 号：R735.7[医药卫生—肿瘤] R730.3[医药卫生—临床医学]