Screening Specific Antibody Of Light Chain Protein Of Botulinum Toxin B And Detecting Its Activity

Botulinum toxin is a polymer protein neurotoxin produced by Clostridium botulinum, is now known the most toxic of a biological toxin in natural and synthetic toxins. As long as it is the inhibition of acetylcholine neurotransmitter release, causing muscle paralysis, a serious cause of death. Botulinum toxin B is named Neurobloc in Europe; it is one type also can lead to food poisoning. At present studies on botulinum toxin B are less, but also a lack of drugs and treatments of it.Phage antibody libraries Tomlinson I + J is built by Cambridge MRC HGMP(Human Genome Mapping) Resource Centre, it has 100 million kinds of different single-chain antibody scFv fragment, large storage capacity, high diversity, in the case of an unknown cloned sequence screened, using these two libraries can be screened to high affinity specific antibodies. Therefore, it will lay a solid foundation for specific drug and antibody preparations of botulinum toxin B.By Inducing expression the light chain protein of botulinum toxin B, we can obtain the protein with a purity up to 90%.After enrichment screening four times through phage antibody libraries Tomlinson I+J, we can get several dozens of positive strains, then IPTG was added to Induced expression antibody proteins.The presence or absence and the size of antibody activity are critical, and the experiment uses a green fluorescent protein labeling to detect activity of the antibody. GFP is green fluorescent protein and a living body reporter protein; it can produce green fluorescent at a wavelength range of blue light excitation. SV(SNAP25-VAMP) gene containing the cleavage site of Bont-B, we constructe SV genes manually,and connect the green fluorescent protein(GFP) gene to the SV gene containing Bont B cleavage site and we obtain a recombinant gene GFP-SV, then express the fusion protein GFP-SV. Finally, the size of the activity of Bont-B antibodies can be determined by intensity changes of fluorescence of the green fluorescent protein.Through phage library screening and detection the activity,3 strains antibodies have been received,the affinity constant respectively is(4.38+0.445)×105L/mol,(5.35+0.903)×105L/mol and(1.146 + 0.525)×106L/mol.