用于肉毒毒素活性测定的特异性绿色荧光融合蛋白的制备
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摘要
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(GFP) containing seven serotypes of botulinum toxin cleavage site(SNAP25-VAMP), express in E. coli, purify the expressed product and apply to the determination of activity of botulinum toxin. Methods According to the sequences of SNAPE25 and VAMP genes in SNARE protein complex and the cleavage sites of various serotypes of botulinum toxin, Bam H Ⅰ-HIS6-SNAP62-Eco RⅠ-VAMP57C-TAAHindⅢ(SV for short)gene was designed, synthesized and inserted into vector p ET-28a-GFP. The constructed recombinant plasmid p ET-28a-GFP-SV was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by DEAE anion exchange chromatography, copper ion metal chelate chromatography and Q anion exchange chromatography, and determined for protein concentration by BCA method and for Bo NT/BL activity by ELISA.Results Restriction analysis and sequencing proved that recombinant plasmid p ET-28a-GFP-SV was constructed correctly.The expressed GFP-SV protein, with a relative molecular mass of about 40 000, main existed in a soluble form, and reached a concentration of 18. 4 μg/μl and a purity of more than 70%. The fluorescent intensity of fusion protein reflected the activity of Bo NT/BL and the neutralizing activity of its antibody. Conclusion The prepared specific GFP containing SV was highly expressed in E. coli, which laid a foundation of further determination of activities of various serotypes of botulinum toxin and the neutralizing activities of their antibodies.
Objective To prepare a specific green fluorescent fusion protein(GFP) containing seven serotypes of botulinum toxin cleavage site(SNAP25-VAMP), express in E. coli, purify the expressed product and apply to the determination of activity of botulinum toxin. Methods According to the sequences of SNAPE25 and VAMP genes in SNARE protein complex and the cleavage sites of various serotypes of botulinum toxin, Bam H Ⅰ-HIS6-SNAP62-Eco RⅠ-VAMP57C-TAAHindⅢ(SV for short)gene was designed, synthesized and inserted into vector p ET-28a-GFP. The constructed recombinant plasmid p ET-28a-GFP-SV was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by DEAE anion exchange chromatography, copper ion metal chelate chromatography and Q anion exchange chromatography, and determined for protein concentration by BCA method and for Bo NT/BL activity by ELISA.Results Restriction analysis and sequencing proved that recombinant plasmid p ET-28a-GFP-SV was constructed correctly.The expressed GFP-SV protein, with a relative molecular mass of about 40 000, main existed in a soluble form, and reached a concentration of 18. 4 μg/μl and a purity of more than 70%. The fluorescent intensity of fusion protein reflected the activity of Bo NT/BL and the neutralizing activity of its antibody. Conclusion The prepared specific GFP containing SV was highly expressed in E. coli, which laid a foundation of further determination of activities of various serotypes of botulinum toxin and the neutralizing activities of their antibodies.
引文
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[2]CHEN Y A,SCHELLER R H.SNARE-mediated membrane fusion[J].Nat Rev Mol Cell Biol,2001,2(2):98-106.
[3]刘全,张西臣.绿色荧光蛋白在寄生虫学研究中的应用[J].中国人兽共患病杂志,2005,21(8):728-730.
[4]WU S K.The discovery and development of the Green Fluorescent Protein[J].Imag Sci Photochem,2009,27(1):69-78.(in Chinese)吴世康.绿色荧光蛋白质(GFP)的发现、表达和发展[J].影像科学与光化学,2009,27(1):69-78.
[5]WIEDENMANN J,OSWALD F,NIENHAUS G U.Fluorescent protein for live cell imaging:opportunities,limitations,and challenges[J].IUBMB Life,2009,61(11):1029-1042.
[6]CRAGGS T D.Green fluorescent protein:structure,folding and chromophore maturation[J].Chem Soc Rev,2009,38(10):2865-2875.
[7]HAO Z Y,HONG S L,CHEN X,et al.Introducing bioorthogonal functionalities into proteins in living cells[J].Acc Chem Res,2011,44(9):742-751.
[8]TANG Y,LI Y B,LIN R Y,et al.Efficient in vitro si RNA delivery and intramuscular gene silencing using PEG-modified PAMAM dendrimers[J].Mol Pharm,2012,9(6):1812-1821.
[9]ANDRIES O,DE FILETTE M,REJMAN J,et al.Comparison of the gene transfer efficiency of m RNA/GL67 and p DNA/GL67 complexes in respiratory cells[J].Mol Pharm,2012,9(8):2136-2145.
[10]PIATKEVICH K D,VERKHUSHA V V.Advances in engineering of fluorescent proteins and photoactivatable proteins with red emission[J].Curr Opin Chem Biol,2010,14(1):23-29.