Matić Slavica, Quaglino Elena, Arata Lucia, Riccardo Federica, Pegoraro Mattia, Vallino Marta, Cavallo Federica, Noris Emanuela
Institute for Sustainable Plant Protection, National Research Council, Torino, Italy.
Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Italy.
Plant Biotechnol J. 2016 Jan;14(1):153-9. doi: 10.1111/pbi.12367. Epub 2015 Apr 10.
The rat ErbB2 (rErbB2) protein is a 185-kDa glycoprotein belonging to the epidermal growth factor-related proteins (ErbB) of receptor tyrosine kinases. Overexpression and mutations of ErbB proteins lead to several malignancies including breast, lung, pancreatic, bladder and ovary carcinomas. ErbB2 is immunogenic and is an ideal candidate for cancer immunotherapy. We investigated the possibility of expressing the extracellular (EC) domain of rErbB2 (653 amino acids, aa) in Nicotiana benthamiana plants, testing the influence of the 23 aa transmembrane (TM) sequence on protein accumulation. Synthetic variants of the rErbB2 gene portion encoding the EC domain, optimized with a human codon usage and either linked to the full TM domain (rErbB2_TM, 676 aa), to a portion of it (rErbB2-pTM, 662 aa), or deprived of it (rErbB2_noTM, 653 aa) were cloned in the pEAQ-HT expression vector as 6X His tag fusions. All rErbB2 variants (72-74.5 kDa) were transiently expressed, but the TM was detrimental for rErbB2 EC accumulation. rERbB2_noTM was the most expressed protein; it was solubilized and purified with Nickel affinity resin. When crude soluble extracts expressing rErbB2_noTM were administered to BALB/c mice, specific rErbB2 immune responses were triggered. A potent antitumour activity was induced when vaccinated mice were challenged with syngeneic transplantable ErbB2(+) mammary carcinoma cells. To our knowledge, this is the first report of expression of rErbB2 in plants and of its efficacy in inducing a protective antitumour immune response, opening interesting perspectives for further immunological testing.
大鼠ErbB2（rErbB2）蛋白是一种185 kDa的糖蛋白，属于受体酪氨酸激酶的表皮生长因子相关蛋白（ErbB）。ErbB蛋白的过表达和突变会导致多种恶性肿瘤，包括乳腺癌、肺癌、胰腺癌、膀胱癌和卵巢癌。ErbB2具有免疫原性，是癌症免疫治疗的理想候选物。我们研究了在本氏烟草植物中表达rErbB2细胞外（EC）结构域（653个氨基酸，aa）的可能性，测试了23个氨基酸的跨膜（TM）序列对蛋白质积累的影响。编码EC结构域的rErbB2基因部分的合成变体，用人密码子使用进行优化，并与完整的TM结构域（rErbB2_TM，676 aa）、其一部分（rErbB2-pTM，662 aa）连接，或缺失该结构域（rErbB2_noTM, 653 aa），作为6X组氨酸标签融合体克隆到pEAQ-HT表达载体中。所有rErbB2变体（72 - 74.5 kDa）均瞬时表达，但TM对rErbB2 EC积累不利。rERbB2_noTM是表达量最高的蛋白质；它用镍亲和树脂溶解并纯化。当将表达rErbB2_noTM的粗可溶性提取物给予BALB/c小鼠时，引发了特异性的rErbB2免疫反应。当用同基因可移植的ErbB2(+)乳腺癌细胞攻击接种疫苗的小鼠时，诱导了强大的抗肿瘤活性。据我们所知，这是rErbB2在植物中表达及其诱导保护性抗肿瘤免疫反应功效的首次报道，为进一步的免疫学测试开辟了有趣的前景。
