为建立肿瘤内皮标志物1(tumor endothelial marker 1， TEM 1)ELISA检测方法，依据TEM 1基因胞外区片段序列及原核表达载体pMAL-p5x多克隆位点设计引物，采用PCR技术从含TEM 1全长基因载体中扩增目的片段，将双酶切的目的基因和载体经胶回收连接后插入表达载体并经测序鉴定，在转入宿主菌后用IPTG诱导目的蛋白表达，用分离纯化后的目的蛋白分别免疫鼠和兔以制备多克隆抗体并用ELISA和流式细胞术对抗体进行鉴定，采用兔抗TEM1为捕获抗体、鼠抗TEM 1为检测抗体建立双抗体夹心ELISA检测方法，棋盘滴定法探索各抗体最佳工作浓度，倍比稀释TEM 1, 检测建立方法的灵敏度。成功构建的重组表达质粒pMAL-p5x-hCD248，经纯化复性后获得目的蛋白，免疫后获得兔多抗抗体效价为1∶1 024 000、鼠多抗抗体1∶512 000，棋盘法确定捕获抗体、检测抗体及酶标抗体最佳浓度分别为1∶500、1∶16 000和1∶20 000，经检测证实该方法测定下限为18.31 ng/mL；检测50例恶性肿瘤患者与50例健康人血清TEM 1含量， 发现差异无统计学意义。

This study aimed to establish an improved ELISA to detect endosialin. The primers were designed based on TEM 1 gene sequence and multiple cloning sites of prokaryotic expression vector pMAL-p5x by primer 5.0. The targeted gene fragments were amplified from the vector containing the whole TEM 1 gene. The amplified fragments and vector pMAL-p5x were digested by relative restriction enzymes and the targetod gene sequence was inserted into pMAL-p5x after gel extraction. Recombinant plasmid was confirmed by sequencing. TEM 1 protein was prepared with expression and purification. Polyclonal antibodies were generated after multiple immunizations with TEM 1 protein, and then were identified and characterized via ELISA and flow cytometry. Double-antibody sandwich ELISA was constructed with the rabbit and mouse polyclonal antibodies. Detection sensitivity was verified by double dilution method with hCD248 protein. The recombinant expression plasmid pMAL-p5x-hCD248 was constructed successfully, hCD248 protein was obtained after purification and renaturation process, valences of the polyclonal antibodies of the rabbits and mice were 1∶1 024 000 and 1∶512 000 respectively, and the optimum concentrations of capturing antibodies, detection antibodies and goat-anti-mouse secondary antibodies were 1∶500, 1∶16 000 and 1∶20 000, respectively. The detection sensitivity of this method was 18.31 ng/mL. We evaluated TEM 1 levels in serum samples from 50 patients with malignant tumors and 50 healthy control individuals, however, there was no difference in the levels of TEM 1 between the two groups.