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目的制备含有7种血清型肉毒毒素切割位点(SNAP25-VAMP)的特异性绿色荧光融合蛋白,在E.coli中诱导表达,纯化后用于肉毒毒素活性测定。方法根据SNARE蛋白复合物中SNAPE25和VAMP基因序列及各血清型肉毒毒素(Bo NTs)的切割位点,设计合成含有SNAREs中突触小体(SNAP25)和突触小泡膜蛋白(VAMP)的Bam HⅠ-HIS6-SNAP62-Eco RⅠ-VAMP57C-TAA-HindⅢ(以下简称为SV)基因,连接至p ET-28a-GFP载体上,构建重组质粒p ET-28a-GFP-SV,转化感受态E.coli BL21(DE3),IPTG诱导表达后,经DEAE阴离子交换层析、Cu2+金属螯合层析、Q阴离子交换层析3步纯化,BCA法测定纯化产物的蛋白浓度,ELISA法检测B型肉毒毒素轻链蛋白(Bo NT/BL)活性。结果构建的质粒p ET-28a-GFP-SV经双酶切及测序鉴定证明构建正确,表达的GFP-SV融合蛋白相对分子量约40 000,主要以可溶形式存在,蛋白浓度为18.4μg/μl,纯度可达70%以上。利用该融合蛋白的荧光强弱变化可测定Bo NT/BL活性大小,同时也可测定其抗体中和活性大小。结论制备的含有SV的特异性绿色荧光融合蛋白在E.coli中获得了高效表达,为后续各型肉毒毒素活性检测及抗体中和毒素活性检测奠定了基础。
Objective To prepare a specific green fluorescent fusion protein containing seven serotype botulinum toxin cleavage sites (SNAP25-VAMP), which was induced in E. coli and purified for botulinum toxin activity determination. Methods According to the sequence of SNAPE25 and VAMP gene in SNARE protein complex and the cleavage site of each serotype botulinum toxin (Bo NTs), we designed and synthesized synaptosomal bodies (SNAP25) and synaptic vesicle membrane protein (VAMP) Bam HⅠ-HIS6-SNAP62-Eco RⅠ-VAMP57C-TAA-HindⅢ (hereinafter referred to as SV) gene was ligated to p ET-28a-GFP vector to construct recombinant plasmid p ET-28a-GFP- After induced by IPTG, E.coli BL21 (DE3) was purified by DEAE anion exchange chromatography, Cu2 + metal chelation chromatography and Q anion exchange chromatography. The protein concentration of the purified product was determined by BCA method. Botulinum toxin light chain protein (Bo NT / BL) activity. Results The constructed recombinant plasmid pET-28a-GFP-SV was verified by double enzyme digestion and sequencing. The constructed recombinant GFP-SV fusion protein has a relative molecular weight of about 40 000 and mainly exists in a soluble form with a protein concentration of 18.4 μg / μl , Purity up to 70%. The fluorescence intensity of the fusion protein can be used to measure the activity of BoNT / BL, and the size of its antibody neutralizing activity can also be determined. Conclusion The specific green fluorescence fusion protein containing SV was highly expressed in E.coli, which lays the foundation for the detection of various botulinum toxin activities and the detection of antibody neutralizing toxin activity.