Production Of Human Monoclonal Antibodies Against Protective Antigen Of Bacillus Anthracis

Anthrax, a potentially fatal infection, is caused by a virulent and highly contagious zooanthropozoonosis named Bacillus anthracis. Bacillus anthracis is one of the greatest threats in biological warfare because anthrax has high attack rate, short latent period, high fatality rate and the anthrax spores can resist environmental degradation. Although antibiotics are effective, these modalities have their limitations. Thus, there exists a need for improved therapies to augment available treatment options for anthrax.. Antibodies are highly specific and effective, making them a logical choice for an anthrax toxintherapeutic. Human antibodies are safe and well tolerated for many therapeutic purposes. Therefore human antibodies to anthrax toxin should have therapeutic value in human anthrax infections. In this study, the domain 4 of the protective antigen of Bacillus anthracis is used as neutral antigen, human neutral antibody is selected from large fully synthetic human antibody libraries. This work lays a foundation for the production of therapeutic human antibody.Firstly, based on the amino acid sequence(P13423,140aa)and the gene sequence(420bp,GenBank Accession:AY428556)of the domain 4 of protective antigen of Bacillus anthracis, its nucleotide sequence was optimizedly designed and.using the method of SOE-PCR, the domain 4 gene of the protective antigen was constructed from twelve chemically synthesized oligonucleotides. The correct domain 4 gene of the protective antigen was verified by sequencing , then it was cloned into the expression vector. The cloned gene was expressed in E.coli jointly with the N1 domain of protein gIII, which is an outer membrane protein of helper phage M13KO7 and the recombinant protein was purified.Secondly, the high purity fusion protein is used as target antigen, the human antibody binding specifically to the target antigen was selected from large fully synthetic human antibody libraries by biopanning.Finally, human antibody gene is expressed in soluble form, and the expressed recombinant scFv was analyzed if it is specifically binding to the domain 4 of the protective antigen.In this study, the domain 4 gene of the protective antigen of Bacillus anthracis was obtained by SOE-PCR. The molecular weight of the fusion protein expressed in E.coli was about 25kD, The domain 4 of the protective antigen of Bacillus anthracis was expressed as soluble protein in a high level in the cytoplasm of E.coli, and the recombinant protein was about 36% of total proteins and the purity was...