| Botulinum neurotoxin (BoNT), which is the most toxic protein, is produced as a potent toxin by the anaerobic clostridium botulinum. It is known as one of the six top bioterrorism agents. The events of food poisoning caused by botulinum toxin have also occurred in recent years and botulinum toxin has also been widely used in cosmetic and the clinical treatment of muscle spasm, excessive glandular secretion and so on. Regardless of product quality control and samples detection, a rapid, sensitive and reliable detect method is needed. There are, therefore, numerous reasons to test for the presence or the relative concentration of botulinum toxins, not only for the medical testing but also for the bio-terrorism.Seven serotypes (A to G) of botulinum toxin are currently known. Each serotype is composed of a heavy and a light chain linked tighter by disulphide bonds. The N-terminal end of the heavy chain is responsible for binding to specific pre-synaptic neuronal cell receptors and the C-terminal end facilitates internalization. Once inside the cell, the light chain transverses the membrane of the endocytolic vesicles and cleaves specific proteins within the cytoplasm essential for the docking and fusion of neurotransmitter containing vesicles at the nerve terminal. Subsequently, the extra-cellular release of neurotransmitter into the neuromuscular junction is blocked and results in a flaccid muscular paralysis. The substrate of type A, E and C1 is synaptosome associated protein SNAP25.Currently, the standard detect method of botulinum toxin is the mouse acute toxicity test. But this method needs a long cycle, complicated steps, requires sacrificing a lot of animals and data fluctuation. Therefore, researchers have been actively looking for an alternative approach. Early detections are mainly based on the antigenicity of toxins, which only response to the biomass associated with the toxin. These can not be a direct response to the biological activity. In recent years, there have been some detect methods based on the protease activity of light chain. One of them is the fluorescence detection; the other is based on the internal antigens of the substrates. Our subject is to establish a biological activity detect method of botulinum toxin type A, which mainly from the latter point of views. The basic principle is to use a detecting antibody to an octapeptide, which buried within the native substrates alpha-helix and then exposed following cleavage by botulinum toxin type A.In this study, botulinum toxin is extremely toxic and the source is limited. And it may pose a potential threat to laboratory personnel in practice. However, the single light chain or heavy chain is not toxic. Therefore, we select individual light chain of botulinum toxin A protein to the establishment the detect method. The light chain gene sequence of botulinum toxin type A has been optimized and synthesized according to the laboratory E. coli codon preference. Then it was inserted into pTIG-Trx to create plasmid pTIG-ALC. The recombinant plasmid was transformed into the E. coli strain BL21 (DE3) pLysS and induced by IPTG at 25℃. The expression protein accounted for 44.6% of total bacterial protein. Soluble protein accounted for 36.8% of total bacterial protein, which yield reached to 294.4 mg/L. Its purity was up to over 99% after purification. The results of SDS-PAGE and Western-blot showed that BoNT/A LC protein had good biological activity. Currently, expression of the high activity, soluble light chain protein successfully has not been reported at home and abroad.The gene of synaptosomal associated protein SNAP25 was obtained using RT-PCR method. Then it is cloned into pTIG-Trx expression plasmid. The recombinant plasmid was transformed into the E. coli strain BL21 (DE3) and induced by IPTG. The protein expressed accounted for 26.2% of total bacterial protein with a yield as high as 115.4 mg/L. Its purity was up to 95% after purification. The results of SDS-PAGE and Western-blot showed that SNAP25 protein could be cleaved by BoNT/A LC protein.We cloned, expressed and purified the SNS protein, which is the specific cleavage product of SNAP25. The protein expressed accounted for 20.3% of total bacterial protein with a yield as high as 203 mg/L. Its purity was up to 95% after purification. And its good reactogenicity is identified by ELISA, which indicated that it can be used as a standard protein for the detect method.BoNT/A cleavage site-specific anti-peptide SNAP25190–197, antibody is raised in New Zealand White rabbits following conjugation to KLH through the Cys residue. Antibody was purified by the Protein A column. Then this antibody is identified that it has a good sensitivity, which is greater than 125 ng/mL and consistent with the conditions for detection of antibodies. Highly specific SNAP25 cleavage site (190–197) detecting antibody is utilized in this study to develop sensitive, highly reproducible endopeptidase immunoassay. And the indicators of the detect method are studied. The best coated concentration of substrate is 3μg/mL, reaction time was 90 min, the detection limit of light chain protein of botulinum toxin type A is 12 fg/mL, the effective detection range of 0.023-24.0 pg/mL, intraplate mean CV of three groups are 5.2%, 5.4%, 3.9% respectively and between plates mean CV is 6.3%, method recoveries ranged between 92.6%-111.5%, the result of the recoveries of botulinum toxin type A LC in serum, milk and glycerin range from 60.0% to 134.3%, indicating that the method is rapid, sensitive, stable, reliable, and can be used for sample testing.In this study, we use BoNT/A LC and SNS as the reference protein for the detect method. Then we calibrate the dose-effect relationship of the BoNT/A LC, SNS reference protein and BoNT/A LD50, which is 1 LD50/mL of botulinum toxin type A is equivalent to 35.0 pmol BoNT/A LC or 38.9 nmol SNS, the detection limit of botulinum toxin type A is 0.004 LD50 /mL, the best detection range from 0.15 to 2.41 LD50/mL; the method recoveries range from 91.6% to 112.9%.The recoveries of botulinum toxin type A in serum, milk and glycerin range from 84.9% to 135.4%.In this study, we successfully established a reliable and sensitive detection method on biological activity of botulinum toxin type A, which is 1-2 orders of magnitude higher than the method in animals and it can meet the needs of quantitative analysis of samples of botulinum toxin type A.The principle of using a detecting antibody to a substrate sequence buried within the native substrates alpha-helix may be further expanded to the detection of other serotypes of botulinum toxin or other specific enzyme cleavage reactions in the future. |
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