Investigation on the expression and antibody response of chimeric protein lfd1-pa4 from bacillus anthracis in mice

Hassan Mirhaj,1 Hosein honari,2,* Ehsan zamani,3

1. Imam Hossein University
2. Imam Hossein University
3. Imam Hossein University

Abstract

Introduction

Anthrax is a zoonotic and severe infectious disease that caused by bacillus anthracis. the pathogenicity of bacillus anthracis is depends on two important factors in the production of toxins and the formation of capsules. the capsule is made of glutamic acid, it has anti-phagocytic properties. bacterial toxin is made from three protein components: protective antigen (pa) (83 kda), lethal factor (lf) (90 kda), and edema factor (ef) 89 kda. the combination of pa and lf (lethal toxin) after intravenous injection causes the death of laboratory animals, and the combination of pa and ef at the injection site creates edema. the pa protein consists of four regions where its carboxyl terminal region (region 4 or pad4), with binding to host cell surface receptors, causes the introduction of lf and ef into the cell. studies have shown that antibodies generated against four region of pa (pad4) are capable of neutralizing anthrax toxin. the purpose of this study was to express the lfd1-pa4 gene of bacillus anthracis in e. coli host and purify the recombinant protein and produce polyclonal antibody in mice that was successfully performed.

Methods

In this experimental study, pet28 + lf vector was used. the pa4 gene was amplified by pcr and cloned in a pgem-teasy vector. the pa4 and pet28 + lf genes were cut by xbai and hindiii enzymes. the ligation was performed by t4 ligase enzyme and transformed into e.coli bl21 de3.lfd1-pa4 gene sequence was made and with the transformation of this gene into e.coli bl21 de3 , process was confirmed by pcr and sequencing techniques .the expression of recombinant protein was evaluated using sds-page and western blotting techniques. after purification of recombinant proteins by column chromatography, mice were injected in four consecutive times. from the second injection (the first reminder), one week after the injection, blood sampling was performed and analyzed by elisa.

Results

In this study, with the design and construction of a gene cassette and transferring to bacteria, the recombinant protein (fusion) was obtained at about 48 kda. by injecting mixed and fusion proteins, elisa results showed that the antibody titers against pa4, lfd1, lfd1-pa4, and mixed lfd1 to pa4 were suitable in mice, and the antibody titer increased with fusion of lfd1 to pa4.

Conclusion

In this study, dual-antitoxin was produced in the form of a chimeric protein lfd1-pa4, which can control the formation of a toxic complex and transfer of the subunit a (lf) to the host cell. the monoclonal antibody against pa is more effective among anti-toxins produced, but compared to the lfd1-pa4 chimeric protein and this antibody, the chimeric protein is preferable to use as a vaccine because the monoclonal antibody against pa in cases where the anthrax toxin genetically engineered or antibody-resistant, it loses its efficacy. for this purpose, the antibody response against the lfd1-pa4 chimeric protein was evaluated by a mixed protein (lfd1 + pa4), which determined that the lfd1-pa4 chimeric protein produced a stronger antibody response. in addition to antibody response against pa(by lfd1-pa4), the presence of lf in the chimeric protein produced more antibody response than the mixed protein (lfd1 + pa4) and this adjuvant effect lies in the lfd1 domain of lf. on the other hand, given the similarity of aminoacid sequences at the amino end of lf and ef (their binding position to pa), antibodies produced can interact with ef and inhibit it. therefore, the production of anti-lf antibodies, in addition to preventing its binding to pa, affects ef binding to pa and forms edema toxin, resulting in an increase in the effectiveness of the chimeric protein lfd1-pa4 as a vaccine. studies by baillie et al. showed that complete protein lf and its one region (lfd1) in the animal challenge with bacillus anthracis sti strain spores provides complete protection . rezaei et al. also showed that the antibody titre produced against lfd1 has high immunogenicity . in this study, the amount of antibody produced against the fusion protein is appropriate and is superior to that of the mixed protein.thus, the fusion of regions 1 of lf and 4 of pa (lfd1-pa4) is more suitable for making the vaccine from the mixed protein (lfd1 + pa4).

Keywords

Anthrax, pa4 ، lfd1 ، lfd1- pa4, antibody titer