Evaluation of a single amino acid substitution at position 79 of human IFN-α2b in interferon-receptor assembly and activity.

Type I interferons (IFNs) are homologous cytokines that bind to a cell surface receptor and establish signaling pathways that motivate immune responses. The purpose of the current study is to assess the activity of a novel-engineered IFN-α2b. The crystallographic structure of IFN-α2b and its receptors was acquired from Protein Data Bank. Various amino acid substitutions were designed based on structural properties and other biological characteristics of residues to find the most effective amino acid on IFN affinity to advanced activities. The IFN-α2b mutants and receptors have been modeled and the interactions between two proteins have been studied as in silico by protein-protein docking for both mutants and native forms. The proper nucleic acid sequence IFN-α2 (T79Q) has been prepared based on the selected mutant. The modified IFN gene was cloned in pcDNA 3.1(-) and introduced to Chinese Hamster Ovary (CHO) cell line. Antiviral and antiproliferative assays of native and IFN-α2 (T79Q) proteins were performed in vitro. The results showed two-fold increasing in IFN-α2 (T79Q) activity (antiviral and antiproliferative activity) in comparison to native IFN-α2b. This engineered IFN-α2b may have significant novel therapeutic applications and in silico studies can be an influential method for practical research function and structure of these molecules.

New Brucella abortus S19 Mutant to Improve Distinction Between Infected and Vaccinated Animals.

BACKGROUND: Using Brucella abortus Strain 19 (S19) to control bovine brucellosis is restricted due to induce antibodies to the O-side chain of the smooth lipopolysaccharide (LPS) which may be difficult to differentiate vaccinated and infected animals. Furthermore, it is virulent for humans and can induce abortion to cattle. OBJECTIVES: The aim of this study was to employ gene knockout B. abortus S19 for the first time to eliminate diagnostic defects and obtain the attenuated mutant strain. MATERIAL AND METHODS: The wbkA gene, which is one of the LPS O-chain coding genes, was knocked out in vaccinal Brucella abortus S19. The proliferative response and immunoglobulin M production were analyzed in wbkA deletion strain-infected BALB/c mice. RESULTS: The loss of wbkA gene function resulted in induction of the splenocyte proliferative response in mice infected by the mutant S19 strain compare to those induced by parental S19 and RB51 strains. Moreover, wbkA mutant did not induce any IgM antibody response using the enzyme-linked immunosorbent assay. CONCLUSIONS: As a result, the new mutant S19 strain had deficiency in its LPS O-chain structure, besides cannot induce IgM response then, reduce mistakes to discriminate between vaccinated and infected animal, and also can be considered as a new vaccine candidate.

Design and characterization of an aequorin-based bacterial biosensor for detection of toluene and related compounds.

An aequorin-based Escherichia coli strain JM109 biosensor was constructed and characterized for its potential to detect toluene and related compounds in aqueous solutions. The biosensor was constructed based on a PGL2 plasmid carrying the lower pathway promoter (Pu) of the xyl operon of Pseudomonas putida mt-2, which was incorporated with transcriptional activator xylR and fused to aequorin cDNA named pGL2-aequorin. Binding of xylR protein to a subset of toluene-like compounds activates transcription at the Pu promoter, thus expression of aequorin is controlled by xylR and Pu. In this work we have compared the effect of Shine-Dalgarno (SD) and T2 rrnß terminator sequence in the expression of aequorin. According to the sensitivity of aequorin and increase in the signal-to-noise ratio, this reporter enzyme has reasonable sensitivity compared with other reporter systems. The results indicate higher expression of aequorin in the presence of SD and T2 rrnß. The activity of aequorin in recombinant whole-cell biosensor was linear from 1 to 500 µm of toluene. The bioluminescence response was specific for toluene-like molecules, so this biosensor cells would be able to detect toluene derivative contamination in environmental samples, accurately.