Background: Cholera as diarrheal illness is one of the most important causes of death and people's disability in different societies. Colonization factor pili (tcpA) and cholera toxin are the most important factors in the pathogenesis of Vibrio Cholera. B subunit of cholera toxin (ctxB) and tcpA have the ability to induce immune responses. The aims of this study was production of CTXB, tcpA, CTXB-tcpA recombinant protein and evaluation of antibody titers against separately, cocktail and chimeric protein in mice. Methods: In this research study, ctxB، tcpA and ctxB-tcpA genes were cloned in pET28a and pET32a vectors. Recombinant plasmids was transformed to Escherichia coli (E. coli) BL21 DE3 and expression was induced with IPTG. The protein expression were evaluated by SDSPAGE and Western Blotting analysis. The recombinant proteins were purified using Ni– NTA affinity chromatography. Mice immunization were done subcutaneously or intraperitoneally. Antibody titer was determined by ELISA in immunized mice sera. Results: SDS PAGE and western blotting confirmed expression and purification of recombinant proteins. The yield of purified CTXB, tcpA, CTXB-tcpA proteins was 15/570, 11/533 and 33/100 mg/L, respectively. ELISA results showed satisfactory immunization of mice. There was no significant difference in antibody titers against CTXB-tcpA protein and CTXB, tcpA cocktail. Also, no significant difference was observed in titers between subcutaneously or intraperitoneal injection. Conclusion: The low differences in the antibody titer may be related to the longevity of memory cells and also their injection method. Due to the advantages of chimeric proteins, CTXB-tcpA protein could be a good alternative instead of protein cocktail to stimulate the immune system.

Background and aims: Vibrio cholerae is a gram-negative bacterial pathogen that causes diarrheal disease. One of the most important factors in the pathogenesis is Vibrio cholera. Pili also is co-regulated with toxin, which is necessary for bacterial colonization in intestine. The aim of this study was to examine recombinant protein expression and tcpA bioinformatics. Methods: tcpA gene was studied for rare Codons existence and gene optimization conducted using bioinformatics software. Primers designed for amplification of synthetic tcpA gene. Synthetic gene was cloned in vector pET32a (+). The recombinant plasmid pET32a (+)-tcpA was transformed to E. coli strain BL21 (DE3). tcpA gene expression was induced by IPTG 1mM. Recombinant protein expression was evaluated using SDS PAGE and Western blotting. Ni-NTA affinity chromatography was used for purification of recombinant protein. Results: Codon adaptability index of naive tcpA gene was 0. 6, while optimized gene had Codon adaptability index of 0. 9. The prevalence ratio Codons increased to 75 percent through Codon optimization. Enzyme analysis approved tcpA gene cloning in pET32a (+) expression vector. A protein with a molecular weight of 38. 6 kD was seen on SDS-PAGE and its reaction with the antihistidine antibodies was confirmed by Western blot. The purified protein amount was 11. 533milligram per liter. Conclusion: Optimization led to the expression of recombinant tcpA. Regarding tcpA antigenicity, this protein is a good candidate to develop immunity against cholera gene.

Background: Vibrio cholerae is a gram-negative bacterial pathogen that causes diarrheal disease cholera. One of the most pathogenic factors of Vibrio cholera is pili. Pili plays an important role in colonization and persistence of bacteria in small intestine.Materials and Methods: In this study, pili A (tcpA) gene was amplified by Polymerase chain reaction (PCR) method and sub-cloned into expression vectors such as pGEX4T-1. Escherichia coli competent cells were transformed by recombinant plasmids and the expression of protein with IPTG.The recombinant proteins were purified by affinity chromatography (GST) and immunoblot analysis was used for evaluation of new recombinant proteins antigenicity. The concentration of recombinant proteins was measured according to Bradford assay.Results: The results of this study indicated that recombinant proteins were expressed successfully in competent cell of E. coli, such as E. coli BL21 (DH3). The recombinant protein was purified by affinity chromatography (GST). The immunoreactivity pattern of anti-Tcp antibody with recombinant proteins of tcpA showed that the recombinant proteins had antigenic properties.Conclusion: Because these recombinant proteins are antigenic, these proteins may be considered as tentative candidates for designing cholera vaccine.

Background & Objectives: tcpA protein and B subunit of cholera toxin are the most important virulence factors of Vibrio cholorea. In the current survey, we applied the C-terminal of clostridium perferingenes toxin as a delivery system to bind the CtxB-tcpA fusion as an antigen, cloning it in a prokaryote vector, evaluated the level of expression. Materials & Methods: In experimental survey, the ability of a constructs based on CtxB-tcpA-C-CPE with complete protein sequences of each protein was studied. After amplification of tcpA, ctxB, and c-cpe genes using PCR, they were cloned in expression plasmids. For fusion protein, all of the three protein sequences were constructed with linker. After expression, the proteins were purified and then confirmed using immunoblot methods. Results: This fusion protein consists of 484 amino acids. PCR amplification for the c-cpe, tcpA and ctxB genes amplified 381, 375 and 675 bp,respectively. The result of enzymatic restrictions and sequencing indicated the exact homology of the synthesized proteins and the others submitted in NCBI. According to the SDS-PAGE results, the tcpA, CtxB, and C-CPE proteins were 15, 25 and 25 kD respectively. Conclusion: According to physicochemical results, this fusion protein may be suitable candidate as a vaccine, however,further experimental trials are needed to approve this conclusion.

Background and Objective: Vibrio is a genus of bacteria that are widely distributed in aquatic environments. The genus includes several important pathogens that endanger farm animals and humans who ingest seafood or water contaminated with the bacteria. Virulence of Vibrio spp. is regulated by ctxAB and tcpA genes. The aim of this study was to determine the prevalence of Vibrio spp., and tcpA and ctxB virulence genes in isolates from surface water and salt water samples collected from Golestan Province, Iran. Methods: Overall, 115 water samples were collected from the Caspian Sea coast, lagoons and rivers in the Golestan Province. The samples were filtered by membrane filtration method, and enriched in alkaline peptone water with 1% NaCl. The isolates were grown on TCBS agar, and identified by biochemical tests. Presence of the tcpA and ctxB virulence genes was investigated by polymerase chain reaction. Results: In this study, Vibrio alginolyticus was the predominant species (38%) isolated from the seawater and surface water samples, followed by Vibrio parahaemolyticus (23%), Vibrio harvei (15%), Vibrio fluvialis (14%) and Vibrio damsela (10%). The virulence genes were not detected in any of the isolates found in the study. Conclusion: This study indicates that V. alginolyticus is the most prevalent Vibrio spp. in surface water and seawater samples collected from the Golestan Province, Iran.

Introduction: Cholera is a highly contagious disease that causes severe diarrhea and dehydration. This study investigated podophyllotoxin, deoxypodophyllotoxin, and ursolic acid as inhibitors of tcpA, ompW, and ctxB genes in Vibrio cholerae. Methods: We obtained the crystallized structure of podophyllotoxin, deoxypodophyllotoxin, and ursolic acid from the PubChem database for use as a ligand. The mm2 method in Chem3D v20. 1. 1. 125 was used to optimize the structure of the ligands. We used AutodackVina v. 1. 2. 0 to evaluate the ligands as inhibitors against the active site of the tcpA, ompW, and ctxB proteins. The output results were analyzed and assessed by BIOVIA Discovery Studio 2016 V16. 1. 0 X64. Results: The reported affinities ranged from-6. 8 and-8. 7 kcal/mol. The highest diversity of links was found in tcpA and ctxB. Hydrogen bonds were established with Threonine (91, 111), Glycine (113, 114, 94), and Alanine (92) of tcpA, indicating the effectiveness of ligands against tcpA. The ligands podophyllotoxin, deoxypodophyllotoxin, and ursolic acid showed a variety of hydrogen bonds against ompW and ctxB, respectively, with Arginine, Isoleucine, Histidine, Glycine, and Glutamine. These results demonstrate the excellent inhibitory effects of the ligands against Vibrio cholerae. Conclusion: Vibrio cholerae plays a crucial role in causing pandemic cholera in humans. The predicted conformations of the ligands in this study showed that podophyllotoxin and deoxypodophyllotoxin have higher inhibitory potential than ursolic acid. Therefore, podophyllotoxin and deoxypodophyllotoxin can be potential agents for further research in developing Anti-Vibrio cholerae drugs.