Selection and identification of single domain antibody fragments from

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Selection and identification of single domain antibody fragments from camel heavy-chain antibodies

青龙偃月刀

从骆驼体内分离到重链抗体后。
验证免疫双驼峰，克隆重链抗体可变区库，进一步筛选得到最小单位的免疫结合子。
能够在原核细胞中很好，很稳定的表达，溶解度很高，和抗原有很高的亲和性。

青龙偃月刀

The Fv containing the two variable domains (VH+VL) of immunoglobulins and engineered fragments thereof (e.g. single chain Fv (scFv) [1] or the disulphide stabilised Fv (dsFv) [2]) have been considered as the smallest antibody fragments with retention of the full antigen binding capacity [3,4]. PCR and the development of powerful panning techniques led to the generation of large libraries of scFvs from which several binders could be selected successfully [5-7]. This strategy to obtain binders for a large number of antigens was a major breakthrough in molecular biology. However, the application of the technique is not straightforward [3]. The cloning of two correctly spliced gene fragments (VH+VL) is a difficult step, generating a representative library is tedious, and the genetic constructs are often unstable in the bacterial host. Also, the expression yield, stability and/or functionality of scFv or dsFv often turn out to be problematic [8]. The work of Ward et al. [9] indicated that binders could be readily selected from a pool of cloned spleen VHs from an immunised mouse. These isolated VH domains are expected to bind antigen in absence of VLs. This approach avoids the introduction of a peptide linker used in the scFv constructs which might create additional problems (e.g. reduced affinity, aggregation or proteolytic cleavage) [10]. However, the insolubility of the isolated VH domains expressed in bacteria, and their reduced antigen binding affinity relative to the original VH-VL combination, posed serious limitations for their use. Reshaping the 'VL side' of the human VH to mimic the variable domain of camel heavy-chain immunoglobulins solved the solubility problem [11]. The camelisation of the human VH was based on the knowledge that Camelidae produce a substantial proportion of their functional immunoglobulins as homodimers of heavy chains, lacking light chains [12]. The variable domains of these heavy-chain antibodies are distinguishable from the classical VH domains because they consistently carry important substitutions of otherwise conserved amino acids located in the region which is normally covered by the VL [13,14]. To distinguish the classical VH domain from the variable domains of the camel heavy-chain antibodies we refer to the latter as VHH.

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Randomisation of the third hypervariable loop, the most important loop for antigen binding, on a 'camelised human VH' led to a synthetic library from which several hapten binders were selected [15]. Unfortunately, no useful binders against proteins (HIV regulatory protein rev, lysozyme) could be retrieved from the synthetic camelised human VH library. To avoid this shortcoming, we immunised a camel with two model antigens and generated a VH H library displayed on phage particles. The camel single domain VH H harbours the original, intact antigen binding site [4] and is expected to react specifically and with high affinity to the antigen so that further affinity and specificity improvement is unnecessary. Indeed stable, soluble, specific antigen binders with valuable structural properties [4,14], and good affinities (in the range of 0.2 XlO8 to 2X108 M_ 1 ) were identified from this library. 2. Materials and methods 2.1. Camel immunisation The serum of dromedary (Camelus dromedarius) was shown to be non-reacting with tetanus toxoid or lysozyme. This dromedary was injected with tetanus toxoid (100 μg) and lysozyme (1 mg) according to standard immunisation protocols. The blood of the immunised animal was collected and the peripheral blood lymphocytes were prepared with Lymphoprep (Nycomed) and stored at —80°C until further use.

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骆驼免疫挑选血清中证明与破伤风类毒素或溶菌酶无反应的骆驼。
按照标准免疫方案，给这只单峰骆驼注射破伤风类毒素(100 μg)和溶菌酶(1 mg)。收集免疫动物的血液，得到的用Lymphoprep (Nycomed)制备的外周血淋巴细胞，储存在-80°C备用。

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mRNA 提取，PCR扩增 mRNA从10^7淋巴细胞中分离mRNA，用作模板扩增cDNA，  peripheral blood lymphocytes was isolated and used as template for cDNA synthesis (Micro-FastTrack Kit and cDNA Cycle Kit, Invitrogen). The 5' part of the immunoglobulin heavy chains was amplified by PCR with two gene-specific primers: VHBACKA6: 5'-GAT GTG CAG CTG CAG GCG TCT GG(A\G) GGA GG-3' and CH2FORTA4: 5'-CGC CAT CAá GGTACC AGT TGA-3'. From these PCR products we reamplifled the VHH gene with VHBACKA4: 5'-CAT GCC ATG ACT CGC GGC CCA GCC GGC CAT GGC CGA (GYT)GT (G\C)CA GCT-3' and VHFOR36: 5'-GG ACT AGT GCG GCC GCG TGA GGA GAC GGT GAC CTG-3' containing Sfil and NotI restriction enzymes sites (underlined). The resulting PCR fragments of 450-520 bp were purified from agarose by Geneclean (Bio 101, Inc.), digested with Sfil and Notl, and purified again by Geneclean.