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

我们之前报道了一个由c-erbB2编码的185 kDa的跨膜糖蛋白（p185）参与了人子宫内膜腺癌的侵袭和转移。在这项研究中，我们研究了编码人c-erbB2蛋白的质粒dna的免疫原活性，以评估使用c-erbB2基因作为子宫内膜腺癌肿瘤疫苗免疫原的可能性。利用pCR Bluescript SK（+）载体初步克隆人类c-erbB2启动子区全长（pCMV-ferbB2）或多个长度的编码质粒，并将其转入pcDNA3载体。通过体外转染3T3 NIH细胞和细胞裂解液的免疫印迹分析，检测c-erbB2蛋白的表达。雌性BALB/c小鼠分别用每种质粒20μg三次刺激双股四头肌，并通过四眶穿刺出血。血清在-20℃保存至化验。利用转染pCMV-ferbB2的3T3 NIH细胞裂解液，采用固相酶联免疫吸附法（ELISA）测定血清对人p185的抗体反应。酶联免疫吸附试验表明，注射所有被测质粒诱导较低的抗体反应，而这些质粒表达p185。因此，我们试图产生编码c-erbB2其他区域的构建体，但我们还没有获得诱导更高抗体反应的质粒。另一方面，我们最近报道了ezrin，一种膜-细胞骨架连接蛋白，参与子宫内膜癌细胞的侵袭能力。在甲基胆色素诱导的小鼠肉瘤中，Ezrin也被鉴定为肿瘤相关移植抗原。因此，我们现在研究利用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.