Moghaddam Mehrdad Moosazadeh, Mousavi Latif, Shokrgozar Mohammad Ali, Amani Jafar, Nazariyan Shahram, Azari Shahram
Applied Biotechnology & Environmental Research Center, Baqiyatallah Medical Science University, Tehran, Iran.
Biologicals. 2010 Jan;38(1):113-9. doi: 10.1016/j.biologicals.2009.04.004. Epub 2009 Dec 14.
An assay for the endopeptidase activities of clostridial neurotoxins in contaminated biotherapeutic products has been developed. Based on a synthetic peptide substrate representing amino acid residues 60-94 of the intracellular vesicle associated membrane protein2 (VAMP2), RT-PCR was used to amplify the VAMP2 sequence. The extended insert was digested with EcoRI and SalI and ligated into pGEX4T-1 vector for construction of the pGEX4T-1/VAMP plasmid for expressing in Escherichia coli a fusion protein linked to glutathione S-transferase (GST). The fusion protein was purified by affinity chromatography and used in an ELISA assay for comparison with the commercially available synthetic VAMP peptide and rabbit polyclonal antiserum. The identity of the immunoreactivity of recombinant VAMP2 protein with the chemically synthesized peptide was demonstrated by western blot. Our results indicated that recombinant VAMP2 peptide not only reacted with specific polyclonal antibody in a dose-dependent manner, without any remarkable difference observed between the reactivity of the fusion protein and commercial VAMP2 segment peptide, but also cleaved by botulinum neurotoxin type B (BONT/B) after endopeptidase assay. Thus, recombinant VAMP2 could serve as a replacement for VAMP2 synthetic peptide, potentially useful in endopeptidase assays for replacement of the currently used mouse bioassay for clostridial neurotoxins contaminating biotherapeutic products.
已开发出一种用于检测受污染生物治疗产品中梭菌神经毒素内肽酶活性的检测方法。基于代表细胞内囊泡相关膜蛋白2（VAMP2）第60 - 94位氨基酸残基的合成肽底物，采用逆转录聚合酶链反应（RT-PCR）扩增VAMP2序列。将扩增后的插入片段用EcoRI和SalI酶切，并连接到pGEX4T-1载体中，构建用于在大肠杆菌中表达与谷胱甘肽S-转移酶（GST）相连的融合蛋白的pGEX4T-1/VAMP质粒。通过亲和层析纯化融合蛋白，并用于酶联免疫吸附测定（ELISA），与市售的合成VAMP肽和兔多克隆抗血清进行比较。通过蛋白质免疫印迹法证实了重组VAMP2蛋白与化学合成肽免疫反应性的一致性。我们的结果表明，重组VAMP2肽不仅能以剂量依赖方式与特异性多克隆抗体反应，融合蛋白与市售VAMP2片段肽的反应性之间未观察到任何显著差异，而且在内肽酶测定后能被B型肉毒杆菌神经毒素（BONT/B）切割。因此，重组VAMP2可作为VAMP2合成肽的替代品，在检测污染生物治疗产品的梭菌神经毒素的内肽酶检测中可能有助于替代目前使用的小鼠生物检测法。
