The Preparation Of Egg Yolk Antibodies Against BoNT/A B And Their Application In Mouse Models

The botulinum neurotoxins (BoNTs) produced by anaerobic bacterium Clostridium botulinum, are most devastating intoxication known, which cause the severe food poisoning named botulism. BoNTs are classified into seven serotypes (A-G) and each type is a dichain polypeptide that consists of a light chain joined by a disulfide bond to a heavy chain. Due to the extreme potency and lethality, therefore, BoNTs are used as important toxin weapons and bio-weapons. The most effective treatment of botulism is injection of antidote in the first24hours. However, equine antidote, which is currently used to treat botulism, is associated with a high incidence of side-effects. It is significant to develop anti-AHc IgY and anti-BHc IgY as an alternative antibody against botulinum neurotoxins.The objective of this reseach is to construct the recombinant AHc and BHc, express them in cytoplasm of E. coli, and evaluate their antigenicity. After successive vaccinations on lying hens with the purified proteins, evaluate the potential protective effect of purified IgY against a high-dose of native BoNTA and BoNT/B.The optimized AHc and BHc gene were synthesized and cloned into prokaryotic expression plasmid pTIG-Trx. Identified by sequencing, the AHc and BHc were synthetized successfully and was cloned into expression plasmids pTIG-Trx, named pTIG-Trx-AHc、 pTIG-Trx-BHc. The resulting recombinant vectors pTIG-Trx-AHc and pTIG-Trx-BHc were transformed into E. coli BL21(DE3) strain, and then were induced by IPTG respectively. Recombinant AHc and BHc (about50kDa) were expressed in E. coli (BL21) and the product were partly soluble. The soluble protein collected were purified by Ni-column affinity chromatography method and the antigenicities of recombinant protein were determined by Western Blot and ELISA (probed with anti-BoNT/B antiserum).The purified AHc and BHc were pre-mixed with an equal volume of Freund’s Complete Adjuvant. The mixtures were given intramuscularly at4-6sites of the pectoral muscle of specific-pathogen-free laying hens. In the same manner, hens were then boosted every2weeks after the first immunization and CFA was substituted with Freund’s Incomplete Adjuvant. The hen’s blood was sampled the day prior to the first immunization and seven days after each injection; furthermore, eggs were collected and labeled accordingly during the experimental period. The eggs for obtaining IgY were collected from50to65days after initial immunization. Under acidic condition, IgY was prepared by dilution method with modification and identified by SDS-PAGE. The purified anti-AHc IgY and anti-BHc IgY both showed two typical protein bands with relative molecular masses of68kDa (heavy chain) and25kDa (light chain). The titer of anti-BHc IgY was at a low level (1:800) after primary immunization and reached the maximum level of105finally. The kinetic of the serum in response to the introduction of BoNT/B were similar to what was observed in the IgY in the yolks.50%lethal dose (LD50) of biologically active BoNT/A and BoNT/B complex were determined in BALB/c mice via intraperitoneal injection and intragastric administration. Protective effect of the anti-AHc IgY by pre-incubation with BoNT/A was evaluated in mouse assay. Three different IgY dilutions (100μg/ml、200μg/ml、400μg/ml) were premixed with the same volume serial BoNT/A (100×LD50、200×LD50、400×LD50) in gelatin phosphate buffer. Incubated at37℃for45min, the mixtures were injected i.p. in the all groups. The number of survived mice in each group was recorded over a period of96h. Then, the protective effect of the anti-BHc IgY by pre-incubation with BoNT/B via two different routes. The LD50/i.p. of BoNT/A and BoNT/B using Karber’s method were3.817ng/kg. and4.363ng/kg. In addition, the LD50/i.g. of BoNT/B was1.135μg/kg, nearly100times of LD50/ip.. The mice that given a dosage of BoNT/B complex displayed typical symptoms of botulism3-6h after challenge, including wasp-waist, ruffled fur, hind limb paralysis and labored breathing.40ng dosage of anti-AHc IgY could protect mice from death caused bymixture injection of20×LD50BoNT/A dose, and anti-BHc IgY experiment got the similar result.To further support the protective effect of anti-BHc IgY against BoNT/B, the treatment activity of IgY was detected in which antibody and toxin were injected via two different routes. Firstly, time and dose dependency of treatment with IgY via i.g. was detected. BALB/c mice were challenged with4×LD50/i.p. and overdose IgY (10mg/kg) was given via i.g. at various timing. After the time dependency experiment, the different dosages of IgY (twofold serial dilutions in GPB) were given to the mice via i.p. route certain timing before or after challenging with a dose of4X LD50/i.g.. Then time and dose dependency of treatment with IgY via i.p. was detected through the same method. In addition, the treatment capacity of IgY injection against death BoNT/B via another route was investigated. According to the data, all the mice survived in groups that were treated i.g. with anti-BHc IgY3h before BoNT/B challenge via i.p. route. Anti-BHc IgY at dosage of4mg/kg showed complete protection following i.p. administration in the mice challenged i.g. with4×LD50/i.p. BoNT/B. From the perspective of botulism prevention, anti-BHc IgY injected i.g. before have the capacity to inactivate BoNT/B for at least4h. In addition, in group that were treated i.p. with anti-BHc IgY before, simultaneously with or1.5h after BoNT/B challenge via i.g. route.In this study, recombinant C-terminal heavy chain BoNT/B and BoNT/A were successfully expressed in E. coli. The two recombinant proteins are dramatically are immunogenic and safe to animals. The soluble AHc and BHc purified by Ni-column affinity chromatography method, were used as the antigen to immunize laying hens for yolk immunoglobulin (IgY) production. The purified anti-AHc IgY and anti-BHc IgY, which pre-incubated with the BoNT/A and BoNT/B, were predominantly involved in the neutralization of BoNT/A toxicity and BoNT/B toxicity respectively. Furthermore, both intraperitoneal and intragastric administration of the anti-BHc IgY could protect mice from death caused by injection of BoNT/B toxin at a lethal dose. Our results therefore suggest that anti-AHc IgY and anti-BHc IgY directed to the He domain are effectively involved in the neutralization and could be considered as preventive and therapeutic intervention in the case of botulism.