抗血清在治疗由蛇咬伤引起的神经毒性中至关重要。然而,有流逝的神经毒素,例如,长链α-神经毒素(也称为长链三指毒素),几乎不被商业消逝的抗蛇毒血清中和;因此,重组的消逝神经毒素可能是一种替代或补充方法,可以改善来自消逝毒液的致死性长链α-神经毒素的抗体产生。这项工作传达了重组长链α-神经毒素的表达,名为HisrLcNTx或rLcNTx,根据报道的最致命的长链α-神经毒素,是从头建造的。合成了rLcNTx的基因,并将其导入表达载体pQE30中,该载体包含用于切除成熟蛋白的蛋白水解裂解区,和他的残基串联用于亲和纯化。将克隆的pQE30/rLcNTx转染到大肠杆菌折纸细胞中以表达rLcNTx。在表达式之后,它是在包涵体中发现的,并折叠成多个Cys-Cys结构同工型。为了观察这些同工型产生针对天然长链α-神经毒素的抗体的能力,用不同的Cys-CysrLcNTx亚型混合物免疫各组兔。体外,体内分析表明,针对不同rLcNTxCys-Cys亚型的兔抗体能够识别纯的天然长链α-神经毒素及其流逝的毒液,但是他们无法中和银环蛇毒素,一种经典的长链α-神经毒素,和其他流逝的毒液。rLcNTxCys-Cys同种型2是在兔中产生最佳中和抗体的免疫原。然而,为了中和黑曼巴Dendroaspispolylepis中流逝的毒液,和珊瑚盾眼镜蛇Aspidelapslubrus,它需要使用两种类型的抗体,使用rLcNTxCys-Cys同种型2产生的抗体和使用短链α-神经毒素产生的抗体。重组消逝神经毒素作为免疫原的表达可能是改善消逝抗血清的替代方法;然而,重组后的神经毒素必须折叠良好才能用作免疫原以获得中和抗体。
Antivenoms are essential in the treatment of the neurotoxicity caused by elapid snakebites. However, there are elapid neurotoxins, e.g., long-chain α-neurotoxins (also known as long-chain three-finger toxins) that are barely neutralized by commercial elapid antivenoms; so, recombinant elapid neurotoxins could be an alternative or complements for improving antibody production against the lethal long-chain α-neurotoxins from elapid venoms. This work communicates the expression of a recombinant long-chain α-
neurotoxin, named HisrLcNTx or rLcNTx, which based on the most lethal long-chain α-neurotoxins reported, was constructed de novo. The gene of rLcNTx was synthesized and introduced into the expression vector pQE30, which contains a proteolytic cleavage region for exscinding the mature protein, and His residues in tandem for affinity purification. The cloned pQE30/rLcNTx was transfected into Escherichia coli Origami cells to express rLcNTx. After expression, it was found in inclusion bodies, and folded in multiple Cys-Cys structural isoforms. To observe the capability of those isoforms to generate antibodies against native long-chain α-neurotoxins, groups of rabbits were immunized with different cocktails of Cys-Cys rLcNTx isoforms. In vitro, and in vivo analyses revealed that rabbit antibodies raised against different rLcNTx Cys-Cys isoforms were able to recognize pure native long-chain α-neurotoxins and their elapid venoms, but they were unable to neutralize bungarotoxin, a classical long-chain α-
neurotoxin, and other elapid venoms. The rLcNTx Cys-Cys isoform 2 was the immunogen that produced the best neutralizing antibodies in rabbits. Yet to neutralize the elapid venoms from the black mamba Dendroaspis polylepis, and the coral shield cobra Aspidelaps lubricus, it was required to use two types of antibodies, the ones produced using rLcNTx Cys-Cys isoform 2 and antibodies produced using short-chain α-neurotoxins. Expression of recombinant elapid neurotoxins as immunogens could be an alternative to improve elapid antivenoms; nevertheless, recombinant elapid neurotoxins must be well-folded to be used as immunogens for obtaining neutralizing antibodies.