Production of Polyclonal Antibody against Domain 4 of Protective Antigen of Bacillus anthracis in Laboratory Animals

Abstract

Anthrax caused by Bacillus anthracis, is an acute infectious disease that most commonly occurs in herbivorous animals and human. The vegetative state of Bacillus anthracis has a triplex exotoxin, which consists of three polypeptide: protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa) and edema factor (EF, 89kDa). PA is considered a primary immunogen for the development of protective immunity against anthrax. This study aims to produce polyclonal antibody in laboratory animals. Domain 4 gene was amplified by PCR from pXOI and inserted into expression plasmid pET28a (+) to create the recombinant pET28a (+)/domain4. The pET28a (+)/domain 4 expression vector confirmed by endonuclease digestion, PCR and sequence analysis. The expression of product domain 4 was confirmed by SDS-PAGE. After induced by IPTG, the expression of product domain 4 was analyzed and then was injected to mice and rabbits. All groups received injection four times in 15 days intervals. Finally, the antibody in animals’ sera was confirmed by ELISA and western blotting tests.