[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

[Cloning, expression and purification of snap-25 proterin]

Clostridial neurotoxin inhibits neurotransmitter release by selective and specific intracellular proteolysis of synaptosomal associated protein of 25KDa [SNAP-25], synaptobrevin/VAMP-2 and syntaxin. SNAP-25 is one of the components that forms docking complex in synaptic ends. This protein is subtrate for botulinum neurotoxins types A,C, and E. Each of these toxin serotypes specifically cleaves SNAP-25 in a particular position and thereby block docking and synaptic vesicle membrane fusion and finally prevents neurotransmitter exocytosis and transition of neurotic signals. Recombinant production of SNAP-25 in the laboratory can be used as a subtrate for the detection of clostridium botulinum types A, and E neurotoxins. In order to use the protein as a subtrate for detection of different types of clostridium neurotoxins in-vitro the protein was produced by recombinant technique. The cDNA from SNAP-25 was synthesized from total RNA purified from frozen Rattus norvegicus brain. and amplified by RT-PCR The amplified fragment was cloned into pET32a expression vector. The identity of recombinant protein was confirmed by Western blot using specific antibody and finally the recombinant protein was purified through an affinity column chromatography [Ni-NTA]. The optimum conditions of expression of SNAP-25 were found to be IPTG[1mM] and incubation at 37°C for 5 hours. The recombinant protein was isolated and purified using Ni-NTA column with imidazole at a concentration of 25OmM. Using enterokinase to cut the fision at 37°C comparatively yielded better results than room temperature. The protein retained its structure during the purification process being suitable for cutting and further tests. The purified protein we obtained can be used as subtrate for detection of clostridium botulinum types A, and E toxins

[Immunological characterization of light chain recombinant protein of clostridium botulinum type A]

Botulinum neurotoxin type A, structurally consists of a 50KD light chain and a 100 KD heavy chain linked by a disulfide bond. The protein can further be divided into three functional domains of which catalytic domain corresponds to the light chain. In this research we aimed to produce recombinant catalytic domain in order to obtain a protective protein. Bacteria were grown in anaerobic conditions and genomic DNA was extracted by alkaline method. Following the gene coding a set of primers was designed and the catalytic domain was amplified through PCR. The PCR product was then cloned into three expression vectors namely pRSETA, pET28a and pET32a. The expressed protein was analyzed on SDS-PAGE and confirmed by ELISA and western blotting and then purified by affinity chromatography. In this research the maximum expression was obtained at 0.5 mM IPTG,OD[600]:0.6 and 15 hours of induction at 30§C. The protein so expressed was purified by affinity coulmn chromatography .The antibody raised against recombinant protein could protect the rats by 100 LD[50]. Though the expression of AT- rich genes in E.coli system is low, we could obtain an appropriate level of expression in this study. The purification of recombinant protein in the early stages was elusive in the extreme by affinity chromatography due to the weak binding of histidine N-terminal to the column, howerer 90% purification was achieved through modification of the technique. The antibody produced against this domain was less protective compared to that of binding domain