Design And Overexpression Of Multi-epitope Tandems Of Botulinum Neurotoxin Type A (BoNT/A) And Its Immunogenicity Analysis

Botulinum neurotoxin (BoNT) is produced by Clostridium botulinum, which is generally recognized as the most poisonous naturally occurring substances. Because it could be easily disseminated by terrorists using an aerosol or could be used to contaminate the food or water supply, the Centers for Disease Control and Prevention and the National Institute of Allergy and Infectious Diseases has classified it as a category A agent. However, there is currently no effective therapeutics for preventing and treating it. Both toxoid vaccines and recombinant vaccines conferred good protection against BoNT intoxication, but resulted in many side effects. Therefore, no vaccine currently to prevent botulism has been licensed. Usually, an optimal vaccine should contain all antigens of seven serotypes, so it is a striving tendency to develop new vaccines of multiepitope tandems against BoNT intoxication.Firstly, based on highly conserved BoNT/A amino acids sequence, B cell epitopes were predicted by analyzing related parameters and its secondary structure. the results showed that the B cell epitops of BoNT/A were probably located at or adjacent to its N-terminal No. 440-450,465-480,538-549,699-710,751-760, 1087-1095, 1224-1231,1263-1270 of the heavy chain of BoNT/A, and N-terminal No. 142-150, 284-292 of the light chain. Then, Combining with a universal T cell epitope as well as the Linker GGS, we designed two multiepitope-tandems of BoNT/A, tandem A and tandem B. All the B cell epitopes of BoNT/A were derived from the epitopes reported and predicted.These two genes was optimized using codon optimization software by replacing rare codons with high-frequency ones according to the inclination for codon usage in E. coli and then the gene fragments were synthesized by overlapping PCR of two steps. As confirmed by sequencing, the two gene fragments were subcloned into the expression vector pQE-30 vector to construct recombinant expression vectors pQE-30A, pQE-30B. The resulting expression vectors pQE-30A, pQE-30B were transformed into E.coli M15[pREP4] competent cells and induced at 37°C for 3 hours with IPTG. Specific expression bands with a relative molecular mass 22.4×10³, 24.9×10³ Da was detected by SDS-PAGE and existed in the form of inclusion body, the target proteins accounted for 70%, 54% of total cell protein, respectively. The expressed protein were one-step purified to homogeneity with 92.2%, 99% of purity using Ni-NTA affinity chromatography method under denatured condition, then renaturated by dialysis. Western blot and indirect ELISA identified its good antigenicity.The renaturated proteins were used as immunogen to inoculate Balb/C mice four times at the dose of 20μg. The blood serum titers of immunized mice were up to 3200 and 10,000, respectively. One week after the last immunization, these mice were challenged i.p. with 2, 5, 10, 20, 40×LD₅₀ of BoNT/A respectively, Four days after challenge, no mice was survived. We analyzed the results and reconstructed another three multiepitope tandems, tandem A', C and tandem peptide.Then tandem A', C and tandem peptide were respectively used as immunogen. The methods of immunization were the same with previous assays. One week after the last immunization, These mice challenged i.p. with 3, 6, and 10×LD₅₀ of BoNT/A, Four days after challenge, no inoculate mice was survived expect those challenged with 3×LD₅₀ of BoNT/A. which was less efficient than the peptide of single epitope. We guess the Linker GGS may not isolate the epitopes effectively, and then resulted in new epitopes which may influence the tandem A, B's ability to protect from BoNT intoxication. In addition, we can conclude that the location of Th could not influence the results greatly. And the denature form of the recombinant proteins can be used as immunogen. These assays laid foundation for further study of multiepitope of botulinum neurotoxin type A.