A Basic Study for Tumor Vaccine Development in Human Endometrial Adenocarcinoma-development of tumor vaccination with plasmid DNA containing fusion gene encoding c-erbB2 protein

我们先前报道了由c-erbB2编码的185 kDa跨膜糖蛋白(P185)参与了子宫内膜腺癌的侵袭和转移。本研究通过检测编码人c-erb B2基因的DNA的免疫原性，探讨c-erb B2基因作为免疫原用于子宫内膜腺癌免疫接种的可能性。用PCR Bluescrip SK(+)载体克隆人c-erbB2的全长或几个长度的启动子片段，然后将其转入pcDNA3载体，构建成编码人c-erbB2全长或几个长度启动子区域的重组载体pCMV-ferbB2。通过3T3 NIH细胞体外转染和细胞裂解产物免疫印迹分析检测c-erbB2蛋白的表达。雌性BALB/c小鼠双侧股四头肌注射20μg，共3次，经四眶穿刺法放血。血清保存于-20℃，直至化验。用pCMV-ferbB2基因转染的3T3 NIH细胞裂解液，用固相酶联免疫吸附试验(EL ISA)检测p185的血清抗体反应。酶联免疫吸附试验结果显示，所有受试表达载体均可诱导较低的抗体应答，而这些表达载体均表达p185。因此，我们试图构建编码c-erbB2其他区域的重组蛋白，但我们还没有获得诱导更高抗体应答的质粒。另一方面，我们最近报道了一种膜-细胞骨架连接蛋白Ezrin，参与了子宫内膜癌细胞的侵袭能力。在甲基胆碱诱导的小鼠肉瘤中，Ezrin也被确定为肿瘤相关移植抗原。因此，我们现在研究使用Ezrin基因作为免疫原的可能性。

We have previously reported that a 185 kDa transmenbrane glycoprotein (p185) coded by c-erbB2 is involved in the invasion and metastasis of human endometrial adenocarcinoma. In this study, we investigated the immunogen activity of plasmid DNAs encoding human c-erbB2 protein in order to evaluate the possibility of using the c-erbB2 gene as an immunogen for tumor vaccination in endometrial adenocarcinoma. The plasmid coding the full-length (pCMV-ferbB2) or the several lengths of the promoter regions of human c-erbB2 was constructed by initially cloning with pCR Bluescript SK(+) vector, and then transferring it into pcDNA3 vector. Expression of c-erbB2 protein was tested by in vitro transfection of 3T3 NIH cells and immunoblot analysis of cells lysate. Female BALB/c mice were boosted in both quadriceps muscles with 20μg of each plasmid threetimes and were bled by tetro-orbital puncture. Serum was stored at -20℃ until assay. Serum antibody responses to human p185 were determined in solid-phase enzyme-linked immunosorbent (ELISA) assay by using a cell lysate of 3T3 NIH cells transfected with pCMV-ferbB2. ELISA assay showed that the injections of all tested plasmids induced lower antibody responses, whereas these plasmids expressed p185. Thus we tried to produce the constructs coding the other regions of c-erbB2, however we have not yet obtained the plasmids which induce higher antibody response.On the other hand, we have recently reported that ezrin, a membrane-cytoskeletal linking protein, is involved in invasive ability of endometrial cancer cells. Ezrin is also identified as tumor-associated transplantation antigen in methylcholanthen-induced murine sarcoma. Thus we now investigate the possibility of using the ezrin gene as an immunogen.