Cloning and Expression of Vp2 Gene of Chicken Anemia Vurus Strain Cux-1 in Lactococcus Lactis Mg1363

In this study, the lactococcal plasmid vector pMG36e, was exploited for the cloning, replication and expression of a viral protein-2 (VP2) gene in the bacterial host, Lactococcus lactis strain MG1363. The VP2 gene (0.65 kb) of chicken anemia virus (CAV) of strain Cuxhaven-l was amplified from viral genomic DNA by polymerase chain reaction (PCR) using the VP2FX and VP2RSai primers (Table 6), cloned into the expression vector pMG36e, and electrotransformed into the L. lactis MG1363 host. Sequencing of the recombinant plasmid pMG36e-VP2 using the Forward and Reverse primers (Table 6), showed 99% homology of the VP2 insert with that of the published VP2 CAY Cux-l sequence. SDS-PAGE and Western blot hybridization of L. lactis (pMG36e-VP2; 4.3 kb), showed no significant protein band of VP2 gene product (24 kDa). However, the gene was transcribed under the P32 promoter of the expression vector pMG36e as shown by a 0.7 kb reverse-transcribed product of the VP2 gene

Implementation and use of a DNA microarray for the investigation of environmental responses of pathogenic Neisseria spp

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Bifunctional lipocalin inhibitor of C5 and leukotriene B4 is protective in experimental immune complex alveolitis

OmCI is an ectoparasite derived bifunctional lipocalin that inhibits complement (C) by binding to C5 and also captures the proinflammatory eicosanoid leukotriene B4 (LTB4). We present the crystal structure of recombinant bacterial bOmCI with and without LTB4 at 2.1 Å resolution, and test the relative contributions of C5 inhibition and LTB4 binding to amelioration of experimental immune complex acute lung injury alveolitis (IC-ALI). The structure shows why OmCI is able to accommodate LTB4, arachidonic acid (AA) and 12-OH hydroxyeicosatetraenoic acid (HETEs), but not cysteinyl leukotrienes, prostaglandins or thromboxanes. It also reveals that only minor structural changes occur when bOmCI is bound to LTB4 rather than palmitoleic acid (C16H30O2) which is the dominant fatty acid in the binding pocket of recombinant bOmCI. In accord with the minor structural changes and use of opposite faces of OmCI for C5 binding and entry of LTB4, the two activities of the protein appear to be entirely independent - since the binding kinetics to LTB4 do not change when bOmCI is bound to C5 and the affinity for C5 and inhibition of C activation is unaltered by binding LTB4. In a mouse model of IC-ALI OmCI inhibited neutrophil recruitment and microvascular damage, and reduced protein exudation in the bronchoalveolar space. To examine the relative importance of OmCIs independent inhibitory activities in IC-ALI, OmCI was saturated with LTB4. The saturated protein was a less potent inhibitor of lung inflammation, though both LTB4 binding and C inhibition were required for maximum effect. The dual activity of OmCI may have advantages over conventional biotherapeutics, such as monoclonal antibodies, which typically target only single components of the immune syste

The Structure of OMCI, a Novel Lipocalin Inhibitor of the Complement System

The complement (C) system is a potent innate immune defence system against parasites. We have recently characterised and expressed OmCI, a 16 kDa protein derived from the soft tick Ornithodoros moubata that specifically binds C5, thereby preventing C activation. The structure of recombinant OmCI determined at 1.9 Å resolution confirms a lipocalin fold and reveals that the protein binds a fatty acid derivative that we have identified by mass spectrometry as ricinoleic acid. We propose that OmCI could sequester one of the fatty acid-derived inflammatory modulators from the host plasma, thereby interfering with the host inflammatory response to the tick bite. Mapping of sequence differences between OmCI and other tick lipocalins with different functions, combined with biochemical investigations of OmCI activity, supports the hypothesis that OmCI acts by preventing interaction with the C5 convertase, rather than by blocking the C5a cleavage site