In Vitro Assay Of Botulinum Neurotoxin Endopeptidase Activity And Its Preliminary Application

Botulinum toxin is the most toxic substances are known to the world, botulismpoisoning incidents have occurred, mainly in the form of food pollution. At present, themain use of botulinum toxin horse serum treatment of botulism, easy to produce theimmune response and side effects, at the same time to enter the toxin effect in nerve cellsis very poor. Therefore the development of small molecule inhibitors can enter the nervecell is the main direction of botulinum toxin treatment of drug development. But there areno small molecule inhibitors suitable for the treatment of botulism, establish theappropriate analysis method is of great significance to accelerate the screening andactivity verification inhibitors. This paper established the analysis method of the twokinds of botulinum endopeptidase activity in vitro, and the use of in vitro screening acompound peptidase activity was significantly inhibited in the analysis, the three part ofthe study.Specific studies are as follows:Part1Establishment and optimization of endopeptidase electrophoresis analysismethodWe established for endopeptidase of botulinum toxin type analysis. The substrate wedesign and construct a more suitable for botulinum toxin analysis of fusion protein SNVP:from N side to C side in the order of SNAP-25residues in the1-206,6histidine residueslinker sequence, VAMP1-94,6histidine tag. The successful construction of engineeringbacteria pET-22b-SNVP/Rosetta （DE3）, the purity of recombinant SNVP was purified byA,95%; B toxin light chain of ALc and BLc in response to its, proved that therecombinant SNVP can be ALc and BLc cleavage activity.To SNVP as the substratebuffer, endopeptidase electrophoresis analysis method based on pH, reaction temperature, and DTT conditions were optimized to get the optimal buffer: Hepes buffer; the optimumpH was6.5-7.5; the optimum reaction temperature was37C; The optimum DTTconcentration for2.5-5mM, the kinetic parameters in ALc cut SNVP substrate obtainedby K_m was6.4±0.4mM and k_cat=39±1.5s^-1; similarly obtained BLc cut SNVPsubstrate K_m and k_cat were5.9±0.2mM and45±1.2s^-1.Part2Establishment and optimization of endopeptidase dual fluorescent reportermolecules based on analytical method.In order to meet the requirements of high throughput screening inhibitor, we designand construct the analysis method of endopeptidase dual fluorescent reporter moleculesbased on. First of all, successfully constructed for dual fluorescent reporter molecules ofdifferent serotype toxin recombinant expression plasmid pET-22b-CYA andpET-22b-CYB; purification purity was about90%of the dual fluorescent reportermolecules CYA and CYB, the CYA and CYB reaction by ALc and BLc, show that CYAcan only be ALc cleavage and cannot be BLc cutting, the same CYB can only berecognized by BLc but not by ALc cutting cutting.The emission wavelength ratiodetection of ALc enzyme CYA （528/485） related to analysis direct change ofstandardization for the changes of CYA and SDS-PAGE analysis of CYA, was linearlyrelated to the results of the two methods （R²=0.96）, that can be used in fluorescencewavelength528and485ratio as the substrate change index, and through standardizationto changes in substrate quantity. Optimization of the analytical method of endopeptidasedual fluorescent reporter molecules on the substrate concentration and enzymeconcentration, dynamic detection, detection time interval duration conditions found: therange of substrates CYA suitable for （0.5-32μM）, and CYB suitable for （0.2-32μM）;suitable dynamic detection interval is2min; the continuous detection of the right time for(30min^-120min). CYA peptidase ALc function with time under the emission wavelengthratios （528/485） ranged between0.5-0.9; CYB peptidase BLc action time emissionwavelength ratio （528/485） ranges between0.5^-1.5. Enzyme kinetics parameters determination of ALc substrates of CYA value of K_m was2.33±0.21μ M and k_cat valuewas5±0.4s^-1; similarly obtained BLc cut CYB substrate K_m and k_cat were17.6±2.6μM and4.16±0.28s^-1.Part3Screening and in vivo activity in vitro validation of botulinum toxin type Binhibitors.Utilizing endopeptidase dual fluorescent reporter molecules based on the analysis toscreen the16nicotinic acid compounds.16compounds in10μg effects of2nM BLcenzymatic substrate to be CYB, the inhibition rate more than20%degree of inhibitionand from big to small are respectively B0, B23, B20, B3, B12, B8, B21, B9, B26, B2,B24, B18, B10, among them B0BLc screening results best inhibition, the inhibitoryrate was95.5±2.9%, followed by B23, the inhibition rate was55.4±2.1%.The inhibitory effect of16compounds were verified by electrophoresis based onBLc endopeptidase assay, electrophoresis results can be seen in which the inhibitoryeffect of B23was best, almost completely inhibited the activity of BLc, followed by B0,for the other compounds to inhibit the effect of weak. Screened compounds on mousebotulism detoxification effect analysis found: B0and absolute lethal dose of B toxinincubated with retroperitoneal approaches the poison attack in200μg,80μg,60μg,40μg when the rates were100%,60%,20%,0%, on the protection of mice in adose-dependent. Compounds with absolute lethal dose of B toxin incubated withretroperitoneal approaches the poison attack, when the compound to a dose of200μg, B0can provide complete protection, on mouse B12, B23and B2480%protection rate.