ipaDgene is amongst key genes in Shigella invasion, known as an antigen. PA20 protein of Bacillus anthracic can be separated from native PA protein during the infection and can be found in the serum of the infected patients and make it possible to diagnose the disease. CTxB, a known immune adjuvant, enhances the immunogenicity and is mainly used for the production of recombinant vaccines as a booster for immunization with antigen. In order to produce a vaccine with the immunogenic properties for the diseases, shigellosis and anthrax, different combinations of threeipaD, PA20 and CTxB genes were studied by bioinformatic tools in terms of their stability and similarity to the native proteins that are essential for their accurate performance. The aim of the present study was to introduce the best combination of the three antigens (ipaD, PA20 and CTxB) such that, when they are fused together, have a minimum effect on their structure and activity. Secondary and tertiary structures of proteins derived from different combinations of two or three proteins of ipaD, PA20 and CTxB were studied by MODELLER and PSIPRED software and the stability and lifespan of the fused genes were surveyed using ProtParam software. Analysis of data obtained from different combinations of ipaD, PA20 and CTxB genes by PSIPRED, ProtParam and MODELLER software showed that the best triple combination was PA20-CTxB-ipaD and the best of dual combinations was PA20-ipaD. When a protein combines with other proteins, the three-dimensional structure and physicochemical characteristics may dramatically change. According to the results of the present study, PA20 showed a strong stability, while ipaD showed a poor stability.Furthermore, all proteins in PA20- CTxB-ipaD combination had maintained their stability and this fusion can be used furthermore for vaccine design.

Background and Objectives: Genus Shigella is one of the main causes of diarrhea in human and Bacillus anthracis is the cause of anthrax (a zoonotic disease). IpaD protein is one of the most important virulence factors of Shigella. The cloning of N-terminal ipaD gene along with pa20 gene that have a role in immunization, can be considered as a recombinant vaccine candidate. In this study, we investigated the expression of the recombinant protein domain a-1 protective antigen (PA20) of Bacillus anthracis and N-terminal ipaD gene of Shigella in E. coli.Methods: In this experimental study, primers were designed for pa20 gene and PCR was performed to amplify this fragment. Then, the amplified fragment was cloned into pGEM-T Easy Vector Systems. The pa20 gene was cut using restriction enzymes EcoRI and XhoI and finally, pa20 gene was fused to ipaD gene. pET28a (+) Vector containing the gene cassette ipaD-pa20 was prepared and transformed into E. coli strain BL21 (DE3), and the expression of gene cassette was studied.Results: In this study, the ipaD-pa20 fusion genes in the expression vector pET28a(+), were confirmed by PCR and enzyme digestion. Also, the produced recombinant protein were confirmed by SDS-PAGE and Western blotting.Conclusion: Considering the detection of PA antigen by PA20 antibody and its apoptosis induction and immunization property of the produced IpaD antigen, it can be proposed as a recombinant vaccine candidate against types of Shigella and Bacillus anthracis.

Introduction: Shigella enterotoxin (STxB) is one of the major virulent factors in Shigella dysenteriae type 1 and E. coli O157:H7, which its immunogenicity, adjuvant and delivery characteristic have been proven. Anthrax is a common disease in humans and animals and identifies domain a-1 antibodies of protective antigen (PA) which is 62% of the PA antigens of Bacillus anthracis. Therefore, the purpose of this study was to investigate the expression of recombinant protein domain a-1 protective antigen (PA20) of Bacillus anthracis with Shigella enterotoxin B subunit (STxB) in E. coli.Methods: In this experimental study, primers of pa 20 gene were designed for replacement in stxB-ipaD gene cassette. PCR was performed in order to amplify the fragment and the amplified fragments were cloned into pGEM-Teasy vector. NdeI and SalI restriction enzymes cut pa20 genes and finally the genes pa20 were fused with genes stxB. PET28a (+) Vector containing the gene cassette pa20-stxB was transformed into E. coli strain BL21 (DE3) and expression of gene cassette was studied.Results: pa20-stxB fused genes were confirmed in the expression vector pET28a (+) by PCR, enzyme digestion and Sequencing. The produced recombinant proteins were confirmed by SDS-PAGE and Western blotting.Conclusion: The findings of the current study revealed that this antigen can be raised as an anti-cancer and recombinant vaccine candidate against types of Shigella, Escherichia coli and Bacillus anthracis which can be due to such factors as identification of antigen(PA) by antibody PA20, its apoptosis induction properties, property of immunogenicity, adjuvant and delivery of STxB protein and high expression levels of Gb3 in human cancer cells.