Showing 3 results for Vibrio Cholera
Hamid Kazemian, Mohammad Najafi-Mosleh, Hamid Abtahi,
Volume 15, Issue 7 (12-2012)
Abstract
Background: Vibrio cholera is an important agent causing cholera in human. The expression of Flagellum and the movement of the bacterium are critical in the colonization and virulence of Vibrio cholera. FlaA gene is one the five genes encoding Flagellin which plays an important role in the activity and movement of the bacterium and its colonization which has a significant role in its immunogenicity. The aim of this study was to express and produce the recombinant FlaA protein in E.coli using Western blot method. Materials and Methods: In this experimental study, FlaA gene was proliferated by PCR method using the specific primers and cloned with BamHI and Xhol in pTz57R/T. Then it was proliferated and sequenced in DH5a vector of E.coli. The cloned FlaA gene was inserted into pGEX-4T-1 vector. The cloned vector was transformed to BL21-DE3 of E. coli and successfully expressed by induction of IPTG. The expressed protein was purified by GST affinity resin. For preparation of the primary antibody, the purified recombinant protein was injected to rats. Western blot assay method was used for determining the antigenicity of the recombinant FlaA. Results: Determination of gene sequencing showed that this gene has been proliferated properly and the antibody used in Western blot verified the production of the recombinant protein. Conclusion: The results of this study demonstrate that FlaA protein is immunogenic and can be evaluated in vaccine designing and as a diagnostic tool for detection of cholera infection.
Somayeh Kiaie, Hamid Abtahi, Mohammad Alikhani, Ghassem Mosayebi,
Volume 15, Issue 7 (12-2012)
Abstract
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.
Abolfazl Morad, Mehdi Zeinoddini,
Volume 23, Issue 6 (11-2020)
Abstract
Background and Aim: In the microbial contamination of food and water, identifying the trace amounts of contaminating bacteria has always been of researchers’ interest and concern. The most frequent approach to resolve this problem is using culture-based methods to increase and enrich bacteria samples; accordingly, it extends the bacterial detection process to several hours or days. One of the smart strategies to solve this problem is the concentration of bacteria using physical methods. The present study aimed to enrich Vibrio cholerae as the most essential water-polluting germs. Accordingly, we used the filtration method and evaluated its function by culture method and two detection approaches of Adenosine Triphosphate (ATP) and PCR assay.
Methods & Materials: A certain concentration of V. Cholerae was artificially added to a specified volume of sterile water. Then, the bacteria were extracted from the medium and filtered using 0.450 µm separable filters. Finally, the performance of the pre- and post-filtration processes was compared using bacterial cell culture (CFU), ATP, and PCR assay with the specific primers for the ompW gene of V. cholerae.
Ethical Considerations: This article is a meta-analysis with no human or animal sample.
Results: The present research results indicated that the applied method presented high efficiency and recovery performance. In other words, samples provided no positive response before filtration in both methods; however, after filtration in isolated and recovered samples, the presence of bacteria was detected in the ATP and PCR methods.
Conclusion: In conclusion, the employed strategy can detect V. cholerae in non-culture and in the shortest time in contaminated water samples.
