[Cloning, bioinformatics study and evaluation expression of gene of enterotoxigenic Escherichia coli CFA/I major subunit [CfaB]]

Background and aims: Enterotoxigenic Escherichia coli [ETEC] is the main cause of diarrhea in children in developing countries and also in travelers to these areas. ETEC attaches to host cells via filamentous bacterial surface structures, known as colonization factors. Epidemiological studies suggest that the prevalence of CFA/I is higher than other colonization factors. CFA expressed by ETEC and so represents an important component of any vaccine. Investigation supposed that CfaB as a major subunit of fimbriae is an appropriate candidate for vaccine preparation. In the present study we investigated cloning and expression of CfaB

Methods: In this descriptive study, information about CfaB gene was obtained from gene bank and appropriate primers were designed accordingly. Genomic PCR reaction was performed and its product [CfaB gene] was cloned into pTZ57R/T cloning vector and then subcloned pET28a expression vector. CfaB gene expression was evaluated

Results: Cloning was confirmed by using restriction enzyme and sequencing. Expression of recombinant protein was determined in different conditions [time, media, and host], but native gene inserted in pET28a was not expressed

Conclusion: Native gene employs tandem rare codon which can reduce the efficiency of expression

[Shigella dysentery stxA mutant [R170L-A231D-G234E] gene design and optimization of recombinant protein expression and purification]

Background and aims: Shigella dysentery is one of the most important human pathogenic intestinal bacteria. Entrance of the shigella toxin into the epithelial cells inhibits protein synthesis leading to cell death. In spite of great investigations on vaccine production against S. dysentery, studying to achieve significant stxA recombinant protein still remains important. The objective of this study was to determine the appropriate mutation loci and designing stxA subunit synthetic gene, its further expression and optimization and ultimately assaying purification method for further immunization studies

Methods: Three mutant stxA gene including [R170L-A231D-G234E] were designed and the synthetic gene in pET28a plasmid was obtained and confirmed by PCR. Thereafter the plasmid was transformed into the host cell E.coli BL21 DE3 after which gene expression was optimized and protein purity assay was then performed

Results: Preliminary studies led to mutant stop gene design, after which it was confirmed by synthetic plasmid and PCR. Expression and optimization were then performed which resulted in large amount of protein inclusion bodies. Purification of inclusion bodies and protein which resulted solubilization was done with a combinatorial method

Conclusion: With regard to the mechanism of shiga toxin effect and favorable mutation design with new arrangement, less toxicity of expressing protein is predicted than previous other mutants, posing a better vaccine candidate