Cloning and expression of Brucella cyclic beta-1, 2 glucan transporter gene [cgt]

Brucellosis is an important cosmopolitan infection disease caused by organisms belonging to the genus Brucella. The cgt gene [cyclic beta-1, 2 glucan transporter gene] is a virulent factor in Brucella genus. The present study was conducted with the aim of cloning and expression of Brucella cgt gene. Brucella melitensis cgt gene was amplified from extracted chromosomal DNA by PCR, then PCR product was cloned into pTZ57R and subcloned into pGEMEX-1 expression vector, then expressed in JM109 E. coli strain. Recombinant protein was confirmed by western blot analysis using patient's serum. The PCR product was cloned in pTZ57R plasmid via T/A cloning method. Recombinant plasmid was digested by Band HI and SacI restriction enzymes, the released band was purified and subcloned into pGEMEX-1 expression vector. Then, sample cells were lysed using lyses buffer and sonicated, then electrophoresed on SDS-PAGE. Protein bands were transferred on nitrocellulose membrane and reacted by patient's serum and detected by FIRP conjugated-anti human antibody. We cloned and expressed Brucella abortus cyclic beta-1, 2-glucan transporter gene [cgt] which is an important agent in brucellosis. Using cgt gene mutant may be an effective way for inhibiting or decreasing the pathogenicity of bacteria

Cloning, expression and purification of truncated Chlamydia trachomatis outer membrane protein 2 [Omp2] and its application in an ELISA assay

Although a simple and direct method does not exist for the detection of chlamydial infections, there are situations in which reliable serological tests, with sensitivity related to a specific antigen, could be helpful. The aim of this study was to clone the first 1100 bp of the C. trachomatis outer membrane protein 2 [omp2] gene in order to prepare a recombinant protein for use in an ELISA system designed to recognize the anti- C. trachomatis antibody in patient sera. The PCR product of the chlamydial omp2 gene was cloned in pBluescript and its first 1100 bp was subcloned in the pQE-30 expression vector and induced by IPTG. The recombinant protein was purified by affinity chromatography and its purity was confirmed by SDS-PAGE, gel diffusion and western blot analyses. The purified protein was coated onto a polystyrene microplate and tested by ELISA using patient serum. We have cloned, over-expressed and purified biologically functional recombinant truncated Omp2 from C. trachomatis for use, as a species-specific recognition antigen, in an ELISA system. In this study we determined a cut-off value of 0.345 for this ELISA system using 55 negative sera and measured six positive sera at dilutions of 1:20-1:2560. As a species-specific recognition antigen, the over-expressed and purified recombinant truncated Omp2 from C. trachomatis performed well in an ELISA system

Production of mutant streptokinase recombinant protein

Streptokinase [SK] is most widely used for treatment of myocardial infarction, however, it is the most expensive thrombolytic agent. A major drawback to SK use is the widespread presence of anti-streptokinase antibodies [Abs]. These Abs cause allergic reactions and neutralize streptokinase therapeutic effects. To produce an engineered variant of streptokinase being functional and less antigenic than the native molecule, we cloned and expressed streptokinase mutant gene lacking the C - terminal 42 amino acids. Recombinant protein was confirmed by western blot analysis with anti T7 monoclonal antibodies. pGEMEX-1 expression vector contains T7 gene 10 protein as fusion protein immediately down stream of T7 promoter and before multiple cloning site, streptokinase mutant gene was cloned after fusion protein. We cloned and expressed mutant streptokinase gene, lacking the C-terminal 42 amino acids. If mut-C42 activity was less affected by neutralizing antibodies compared with native streptokinase, this engineered variant could be a preferred alternative to native streptokinase for thrombolytic therapy