| 摘要: |
| 为了制备重组抗A型肉毒毒素（botulinum neurotoxin A，BoNT/A）人鼠嵌合单克隆抗体并分析中和活性，以BoNT/A作为抗原筛选鼠源抗BoNT/A噬菌体单链抗体库，将筛选得到的鼠源抗BoNT/A特异性单链抗体的重链和轻链可变区分别克隆到pGP5KCγ和pGP5KCκ载体构建表达质粒。表达质粒共转染HEK-293EBNA1细胞，瞬时表达抗BoNT/A人鼠嵌合单克隆抗体（monoclonal antibody, mAb）。蛋白A亲和纯化mAb，分子排阻高效液相色谱（size exclusion high performance liquid chromatography，SEC-HPLC）分析mAb纯度，小鼠中和试验评价mAb中和活性。结果显示，经过筛选得到4个序列正确且各不相同的抗BoNT/A单链抗体。4个抗BoNT/A人鼠嵌合mAb表达质粒均构建成功并转染HEK-293EBNA1细胞，根据转染效率指示绿色荧光蛋白的表达推测，50%~62.5%的细胞表达抗BoNT/A抗体。表达上清经过蛋白A亲和纯化，浓度均>200 μg/mL，单体纯度均>90%。小鼠中和试验显示mA1、mA2、mA3和mA4单株抗体均不能完全中和4 LD50的A型肉毒毒素，但从延迟小鼠生存时间可以得出4个抗体的中和活性为mA3>mA2>mA1>mA4。mA1、mA2、mA4中任何一个抗体与mA3连用中和活性为80 LD50/mg。本研究制备了4个具有不同程度中和活性的抗BoNT/A人鼠嵌合mAb，为A型肉毒神经毒素鸡尾酒疗法奠定了基础。 |
| 关键词: A型肉毒毒素 人鼠嵌合 瞬时表达 中和活性 |
| DOI：10.14188/j.ajsh.2020.04.010 |
| 分类号:R378.8+3 |
| 基金项目:兰州市科技重大专项（2017-2-1）；甘肃省科技重大专项（1502FKDA008） |
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| Transient expression and neutralization activity of recombinant anti-botulinum neurotoxin type A human and mouse chimeric monoclonal antibody |
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QIN Haiyan1, NAN Jianjun1, XIONG Ying1, WANG Jianfeng1, CHEN Jijun1
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Fourth Department of Research, Lanzhou Institute of Biological Products Co., Ltd., Lanzhou 730046, Gansu, China
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| Abstract: |
| To prepare recombinant anti-botulinum neurotoxin type A (BoNT/A) human and mouse chimeric monoclonal antibody and analyze the neutralization activity, BoNT/A was used as antigen for screening murine anti-BoNT/A single-chain variable fragment of antibody (ScFv) phage display library, and the variable regions of heavy chain and light chain of murine ScFv were cloned into pGP5KCγ and pGP5KCκ vectors to construct expression plasmid respectively. The expression plasmids were co-transfected into HEK-293EBNA1 cells to expressed anti-BoNT/A human and mouse chimeric monoclonal antibody. The monoclonal antibody was affinity purified using protein A. The purity and neutralization activity were analyzed using size exclusion high performance liquid chromatography (SEC-HPLC) and mouse neutralization test respectively. Due to screening, 4 anti-BoNT/A ScFv with correct and different sequences were obtained. Expression plasmids of the 4 human and mouse chimeric antibody were successfully constructed and transfected into HEK-293EBNA1 cells. According to the expression of green fluorescent protein which was used as transfection efficiency indicating protein, it was speculated that 50%~62.5% of the cells expressed anti-BoNT/A antibodies. The supernatant was affinity purified by protein A. The purified antibody concentration was more than 200 μg/mL, and the monomer purity was more than 90%. Mouse neutralization tests showed that single mA1, mA2, mA3, and mA4 could not completely neutralize 4 LD50 of BoNT/A, but the neutralization activity was mA3>mA2>mA1>mA4 according to prolonged mouse survival time. The neutralization activity of any of the mA1, mA2 and mA4 in combination with mA3 was 80 LD50/mg. Four anti-BoNT/A human mouse chimeric monoclonal antibodies with varying degrees of neutralization activity were prepared, laying the foundation for BoNT/A cocktail therapy. |
| Key words: botulinum neurotoxin A human and mouse chimera transient expression neutralization activity |
