摘要目的 构建小鼠成纤维活化蛋白(FAP)基因的真核表达载体,并检测其在人胚肾细胞及小鼠体内的表达.方法 根据Gene Bank中mFAP基因(NM_007986)全序列设计聚合酶链反应(PCR)引物,获得其开放式阅读框(ORF).将目的基因片段克隆至真核表达载体pcDNA6/myc-His-B,转化并筛选.将重组质粒注射入小鼠尾静脉,在注射后1、3、5、7 d抽提小鼠腓肠肌组织的总RNA,检测FAP表达.结果 经过酶切鉴定、测序比对,确定所筛选的阳性克隆为pcDNA6-mFAP重组子,其可在真核细胞及小鼠体内正常表达,并产生相应蛋白质产物.在小鼠体内注射后第5天,重组子的基因(0.841±0.040)和蛋白表达量(85.380±4.425)%最高,和空白对照组比较差异有统计学意义(P<0.01).结论 成功构建FAP真核表达载体,为研究该基因产物的功能和进行疫苗抗肿瘤实验提供基础.

abstractsObjective To construct a eukaryotic expression system of fibroblast activation protein (FAP) gene and detect its expression. Methods The polymerase chain reaction (PCR) primer was de-signed based on the full sequence of mFAP in GenBank (NM_007986). The mFAP gene was inserted into the eukaryotic expression vector pcDNA6/myc-His-B. The total RNA was extracted and re-transdueted into cDNA from the gastrocnemius on the 1st, 3rd, 5th and 7th day after the vector was inoculated into the tail vein of BALB/C mice. Results The results showed that the positive cloning of mFAP recombinant could express the FAP protein accurately and stably in the eukaryotes, and the biggest amount of FAP gene (0.841±0.040) and protein (85.380 ±4.425)% expression in BALB/C mice appeared on the 5th day, which was significant differ-ence from blank control group (P < 0. 01 ). Conclusion The recombinant eukaryotic expression system has been constructed successfully and accurately.