| Botulinum neurotoxins (BoNTs), considered as the most toxic substances in nature, are produced by the gram-positive and endospore-forming anaerobic bacteria, Clostridium botulinum. These toxins can be divided into seven serotypes (A–G) which are structurally similar, yet antigenically distinct. The alpha and epsilon toxins are 2 of the 4 major lethal toxins of the pathogen Clostridium perfringens. Alpha-toxin (α-toxin), produced by all types of C. perfringens, is a very important pathogenic factor with the biological activities of phospholipase C (PLC) and sphingomyelinase as well as hemolysis, lethality, and dermonecrosis. It can cause gas gangrene, which is a life-threatening infection that presents with fever, pain, edema, myonecrosis, and gas production. The epsilon toxin (ε-toxin) is produced by type B and D strains and composed of 311 amino acids. Theε-toxin is a potent neurotoxin and can lead to fatal illness (enterotoxemia) in a variety of livestock animals, most frequently in sheep. BoNT, Clostridium perfringensαandεtoxins pose major bioweapon or bioterrorism threats. These toxins are listed for Export Control of Dual-Use Biological Agents in many countries. These purified toxins need to be used for toxins detection and vaccine development. Therefore, it is necessary to develop reference materials of important toxins, which will have important significance in military field and scientific value in relative experiments.In this study, firstly, a new purification process was developed for purifying type B neurotoxin. The kinetics of C.botulinum strain growth and neurotoxin production were determined for maximum yield of toxin, and TPOM was the more suitable culture medium, and the strains were cultured in TPOM for 96 hours at 30°C. The neurotoxin was purified by polyethylene glycol (PEG) precipitation and chromatography. Based on the design of full factorial experiment, 20% (w/v) PEG-6000, 4℃, pH 5.0 and 0.3 M NaCl were optimal conditions to obtain a high recovery rate for the type B neurotoxin complex. The following purification of neurotoxin was accomplished by two chromatography techniques using Sephacryl? S-100 and phenyl HP columns. The BoNT/B complex was eluted using 0.6 M of ammonium sulphate, pH5.8. The BoNT/B was eluted at an ammonium sulfate concentration between 0.8 and 0.6 M, pH8.5. The purified BoNT/B was analyzed for its purity using SDS–PAGE and coomassie blue staining, and determined to represent 95% of the total proteins in densitometric quantification of the lane by using BandScan software. The purified BoNT/B concentration was determined to be 0.55 mg/ml using Bradford protein assays, bicinchoninic acid (BCA) assays and UV assays; the BoNT/B concentration in the sophisticated samples was determined using ELISA. The intraperitoneal mouse bioassay was used to test the toxicity of purified the BoNT/B complex. The LD50 of BoNT/B complex using Karber's method was 4.095 ng/kg with a 95% confidence limit between 3.077 and 5.468 ng/kg. These results confirmed the toxicity of BoNT/B.Secondly, the recombinant Clostridium perfringensαtoxin was obtained as the souble forms, and then devopled as the reference material. Briefly, the cpa gene (without the N-terminal signal peptide gene) encoding rCPA was amplified by PCR using the recombinant plasmid pQE30-cpa as template. The PCR product was digested with EcoR I and Hind III, and the excised fragment was cloned into the suitably digested plasmid pTIG-Trx to create the recombinant vector pTIG-cpa. The nucleotide sequences of the cloned cpa genes were confirmed by sequencing. The correct clone vector pTIG-cpa was transformed into E. coli competent cells (BL21-DE3). The rCPA protein was mainly expressed in the soluble form at 16 oC after induction with 0.3 mM of IPTG. The toxin was eluted from a Ni2+-chelating HP column by using an imidazole gradient of 150 mM~250 mM. Further purification was accomplished by size-exclusion chromatography using a Superdex 75-pg column. The purified recombinant protein was analyzed for its purity using SDS–PAGE and RP-HPLC; this protein was determined to represent 99% of the total proteins. The protein concentration was determined to be 0.955 mg/ml using Bradford protein assays, bicinchoninic acid (BCA) assays and UV assays. The N-terminal amino acid sequences of the purified recombinant alpha toxin were determined to be M-W-D-G-K-I-D-G-T-G-T-H-A-M-I, and are consistent with the theoretical sequences. In vitro rCPA activity was detected by determining its effect on egg yolk lipoproteins (increase in turbidity) or murine erythrocytes (hemolysis). The results of these biological assays showed that rCPA is biologically active. Results of homogeneity and stability tests showed thatαtoxin had preferable homogeneity and stability with confidence coefficient of 95% by using ANOVA (analysis of variance).In addition, the truncated etx gene encoding the bioactive rETX protein was amplified by PCR using the pET-his-etx (from our lab) as a template. The truncated etx PCR product was digested with EcoR I and Hind III, and the excised fragment was cloned into the suitably digested plasmid pTIG-Trx to create the recombinant vector pTIG-etx. This vector was identificated by PCR, digestion and sequencing. Then the correct clone vector pTIG-etx was transformed into E. coli competent cells (BL21-DE3). The rETX was mainly expressed in their soluble forms at 16 oC after treatment with 0.2 mM. The toxin was eluted from a Ni2+-chelating HP column by using an imidazole gradient of 170 mM~250 mM. Further purification was accomplished by size-exclusion chromatography using a Superdex 75-pg column. The purified recombinant protein was analyzed for their purity using SDS–PAGE and RP-HPLC; this protein was determined to represent 99% of the total proteins. The protein concentration was determined to be 0.864 mg/ml using Bradford protein assays, bicinchoninic acid (BCA) assays and UV assays. The N-terminal amino acid sequences of the purified recombinant epsilon toxin were determined to be M-K-A-S-Y-D-N-V-D-T-L-I-E-K-G, and are consistent with the theoretical sequences. The cytotoxicity of rETX was determined using a MDCK cell culture model followed by staining of the cells with the metabolic indicator MTS. The CT50 was calculated and determined to be about 80 ng/ml. The result showed that rETX is biologically active. Results of homogeneity and stability tests showed that rETX had preferable homogeneity and stability with confidence coefficient of 95% by using ANOVA (analysis of variance).In conclusion, a new rapid purification process was developed in this study for highly purifying BoNT/B by PEG precipitation and chromatography, and the purified and bioactive BoNT/B was obtained; the purification processes of the recombinant Clostridium perfringensαandεtoxins were developed, and these purified and bioactive toxins were obtained as the souble forms. Then the purity, quantitation, and bioactivities determinations were carried out, and the homogeneity and stability tests were analyzed for the study of the toxin reference materials. In a word, here these results have provided basis for the reference materials development of Clostridium perfringensαandεtoxins and botulinum neurotoxin type B. |
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