Maass David R, Sepulveda Jorge, Pernthaner Anton, Shoemaker Charles B
School of Biological Sciences, Victoria University, Wellington, New Zealand.
J Immunol Methods. 2007 Jul 31;324(1-2):13-25. doi: 10.1016/j.jim.2007.04.008. Epub 2007 May 15.
Recombinant single domain antibody fragments (VHHs) that derive from the unusual camelid heavy chain only IgG class (HCAbs) have many favourable properties compared with single-chain antibodies prepared from conventional IgG. As a result, VHHs have become widely used as binding reagents and are beginning to show potential as therapeutic agents. To date, the source of VHH genetic material has been camels and llamas despite their large size and limited availability. Here we demonstrate that the smaller, more tractable and widely available alpaca is an excellent source of VHH coding DNA. Alpaca sera IgG consists of about 50% HCAbs, mostly of the short-hinge variety. Sequencing of DNA encoding more than 50 random VHH and hinge domains permitted the design of PCR primers that will amplify virtually all alpaca VHH coding DNAs for phage display library construction. Alpacas were immunized with ovine tumour necrosis factor alpha (TNFalpha) and a VHH phage display library was prepared from a lymph node that drains the sites of immunizations and successfully employed in the isolation of VHHs that bind and neutralize ovine TNFalpha.
源自独特的仅重链骆驼科动物免疫球蛋白类（HCAbs）的重组单域抗体片段（VHHs）与由传统免疫球蛋白制备的单链抗体相比具有许多有利特性。因此，VHHs已被广泛用作结合试剂，并开始展现出作为治疗剂的潜力。迄今为止，尽管骆驼和羊驼体型较大且数量有限，但VHH遗传物质的来源一直是骆驼和羊驼。在此我们证明，体型较小、更易于处理且数量丰富的羊驼是VHH编码DNA的优良来源。羊驼血清免疫球蛋白约50%由HCAbs组成，大多为短铰链类型。对编码50多个随机VHH和铰链结构域的DNA进行测序后设计出了PCR引物，这些引物几乎能扩增所有用于构建噬菌体展示文库的羊驼VHH编码DNA。用羊肿瘤坏死因子α（TNFα）免疫羊驼，并从免疫部位引流的淋巴结制备了一个VHH噬菌体展示文库，该文库成功用于分离能结合并中和羊TNFα的VHHs。
