Evaluation of immune response to recombinant Bacillus anthracis LFD1-PA4 chimeric protein

Document Type : Full paper (Original article)


1 Ph.D. Student in Nano Biotechnology, Department of Biology, Faculty of Basic Science, Imam Hossein University, Tehran, Iran

2 Department of Biology, Faculty of Basic Science, Imam Hossein University, Tehran, Iran

3 MSc Student in Cellular and Molecular Biology, Department of Biology, Faculty of Basic Science, Imam Hossein University, Tehran, Iran


Background: Anthrax is a particularly dangerous infectious disease that affects humans and livestock. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. Domains 4 and 1 of the protective antigen (PA) and lethal factor (LF), respectively, have very high antigenic properties. Aims: In this experimental study, the pET28a-lfD1-pa4 expression vector was designed, constructed and transferred into E. coli BL21 (DE3) plysS. Methods: For this purpose, pa4 gene was amplified by polymerase chain reaction (PCR) and cloned in a pGEM T-easy vector. The pGEM-pa4 and pGEM-lfD1 were digested by XbaI and HindIII enzymes. The ligation reaction was performed by ligase T4 enzyme and the gene cassette, lfD1-pa4, was subcloned in pET28a and transferred to E. coli BL21 (DE3) PlysS. Expression and purification of chimeric proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting techniques. The chimera LFD1-PA4 and mixed LFD1+PA4 proteins were injected four times into mice and antibody production was relativity evaluated by enzyme-linked immunosorbent assay (ELISA) test. Results: The results showed that both chimeric and mixed proteins are immunogenic, but LFD1-PA4 has a higher potential to stimulate mice immune system. Conclusion: LFD1-PA4 chimeric protein induced a higher immune response than LFD1+PA4 mixed protein and elicited antibody responses to LF and edema factor (EF), therefore, it holds promise to be a more effective trivalent vaccine candidate to use in anthrax prevention.


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