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Use of fusion protein constructs to generate potent immunotherapy and protection against scorpion toxins

机译：融合蛋白构建体用于产生有效的免疫疗法和针对蝎子毒素的保护作用的用途

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We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for immunisation in rabbits and mice: the aim was to produce in these animal models protective antisera against the most lethal a-type toxins in the venom from the North African scorpion Androctonus australis. The cDNAs encoding AaH I, AaH II and AaH III (the three major alpha -type toxins acting on voltage-sensitive sodium channels) were fused to the sequence encoding the maltose binding protein (MBP). The constructs (MBP-AaH I, MBP-AaH II, MBP-AaH I + II and MBP-AaH III) were expressed in Escherichia coli, and resulting fusion proteins were translocated to the periplasmic space. The recombinant fusion proteins were characterised and used as antigens to generate antibodies in rabbits. These antibodies raised specifically recognised their corresponding radiolabelled-toxin with affinities in the 0.1 nM range. In vitro neutralisation assays indicated that I ml of serum raised against a mixture of fusion proteins was able to neutralise 15 LD50 of the toxic fraction (AaH-G50) purified from the crude venom by molecular filtration through Sephadex G50. In vivo, the fusion proteins induced a long-term protection in mice against the lethal effects of AaH-G50 or of the native toxins. Ten weeks after the beginning of the immunisation programme, mice were challenged with various toxins or AaH-G50 doses. Mice were fully protected against three LD50 of AaH-G50. Our work shows that fusion protein constructs can be used as a vaccine providing efficient immune protection against A. australis venom.
机译：我们报道了重组蝎子毒素以融合蛋白的形式作为抗原用于兔和小鼠的免疫接种：目的是在这些动物模型中产生针对北非蝎子毒液中最具致命性的A型毒素的保护性抗血清澳洲Androctonus。将编码AaH I，AaH II和AaH III（作用于电压敏感钠通道的三种主要α型毒素）的cDNA与编码麦芽糖结合蛋白（MBP）的序列融合。该构建体（MBP-AaH I，MBP-AaH II，MBP-AaH I + II和MBP-AaH III）在大肠杆菌中表达，所得融合蛋白转移到周质空间。对重组融合蛋白进行了表征，并用作抗原以在兔中产生抗体。这些抗体产生的特异性识别其对应的放射性标记毒素的亲和力在0.1 nM范围内。体外中和试验表明，针对融合蛋白混合物的1 ml血清能够通过Sephadex G50分子过滤，中和从粗毒液中纯化的15 LD50毒性级分（AaH-G50）。在体内，融合蛋白诱导小鼠长期抵抗AaH-G50或天然毒素的致死作用。免疫程序开始十周后，用各种毒素或AaH-G50剂量攻击小鼠。充分保护了小鼠免受AaH-G50的三个LD50的侵害。我们的工作表明，融合蛋白构建体可以用作疫苗，从而提供针对澳大利亚南方蛇毒的有效免疫保护。

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