Bornert Olivier, Kocher Thomas, Gretzmeier Christine, Liemberger Bernadette, Hainzl Stefan, Koller Ulrich, Nyström Alexander
Department of Dermatology, Medical Faculty, Medical Center - University of Freiburg, Freiburg, Germany.
EB House Austria, Research Program for Molecular Therapy of Genodermatoses, Department of Dermatology and Allergology, University Hospital of the Paracelsus Medical University Salzburg, 5020 Salzburg, Austria.
Matrix Biol Plus. 2019 Nov 20;4:100017. doi: 10.1016/j.mbplus.2019.100017. eCollection 2019 Nov.
High conservation of extracellular matrix proteins often makes the generation of potent species-specific antibodies challenging. For collagen VII there is a particular preclinical interest in the ability to discriminate between human and murine collagen VII. Deficiency of collagen VII causes dystrophic epidermolysis bullosa (DEB) - a genetic skin blistering disease, which in its most severe forms is highly debilitating. Advances in gene and cell therapy approaches have made curative therapies for genetic diseases a realistic possibility. DEB is one disorder for which substantial progress has been made toward curative therapies and improved management of the disease. However, to increase their efficacy further preclinical studies are needed. The early neonatal lethality of complete collagen VII deficient mice, have led researches to resort to using models maintaining residual collagen VII expression or grafting of DEB model skin on wild-type mice for preclinical therapy studies. These approaches are challenged by collagen VII expression by the murine host. Thus, the ability to selectively visualize human and murine collagen VII would be a substantial advantage. Here, we describe a novel resource toward this end. By immunization with homologous peptides we generated rabbit polyclonal antibodies that recognize either human or murine collagen VII. Testing on additional species, including rat, sheep, dog, and pig, combined sequence alignment and peptide competition binding assays enabled identification of the major antisera recognizing epitopes. The species-specificity was maintained after denaturation and the antibodies allowed us to simultaneously, specifically visualize human and murine collagen VII .
细胞外基质蛋白的高度保守性常常使得产生有效的种属特异性抗体具有挑战性。对于Ⅶ型胶原蛋白而言，区分人类和小鼠Ⅶ型胶原蛋白的能力在临床前研究中具有特别的意义。Ⅶ型胶原蛋白缺乏会导致营养不良性大疱性表皮松解症（DEB）——一种遗传性皮肤水疱病，其最严重的形式会使人极度虚弱。基因和细胞治疗方法的进展使治愈遗传性疾病成为一种现实的可能性。DEB是一种在治愈性治疗和疾病管理改善方面已取得重大进展的疾病。然而，为了进一步提高其疗效，还需要进行临床前研究。完全缺乏Ⅶ型胶原蛋白的小鼠在新生儿早期会死亡，这使得研究人员不得不采用维持残余Ⅶ型胶原蛋白表达的模型，或者将DEB模型皮肤移植到野生型小鼠身上进行临床前治疗研究。这些方法受到小鼠宿主Ⅶ型胶原蛋白表达的挑战。因此，能够选择性地可视化人类和小鼠Ⅶ型胶原蛋白将具有很大的优势。在此，我们描述了一种为此目的的新型资源。通过用同源肽免疫，我们产生了识别人类或小鼠Ⅶ型胶原蛋白的兔多克隆抗体。在包括大鼠、绵羊、狗和猪在内的其他物种上进行测试，结合序列比对和肽竞争结合试验，能够鉴定出识别表位的主要抗血清。变性后仍保持种属特异性，并且这些抗体使我们能够同时特异性地可视化人类和小鼠Ⅶ型胶原蛋白。
