Production of recombinant non-structural protein-3 hydrophobic domain deletion (NS3ΔHD) protein of bluetongue virus from prokaryotic expression system as an efficient diagnostic reagent.

Serological diagnostics for bluetongue (BT), which is an infectious, non-contagious and arthropod-borne virus disease of ruminants, are primarily dependent on availability of high quality native or recombinant antigen(s) based on either structural/non-structural proteins in sufficient quantity. Non-structural proteins (NS1-NS4) of BT virus are presumed candidate antigens in development of DIVA diagnostics. In the present study, NS3 fusion gene encoding for NS3 protein containing the N- and C-termini with a deletion of two hydrophobic domains (118A to S141 aa and 162S to A182 aa) and intervening variable central domain (142D to K161 aa) of bluetongue virus 23 was constructed, cloned and over-expressed using prokaryotic expression system. The recombinant NS3ΔHD fusion protein (∼38 kDa) including hexa-histidine tag on its both termini was found to be non-cytotoxic to recombinant Escherichia coli cells and purified by affinity chromatography. The purified rNS3ΔHD fusion protein was found to efficiently detect BTV-NS3 specific antibodies in indirect-ELISA format with diagnostic sensitivity (DSn = 94.4%) and specificity (DSp = 93.9%). The study indicated the potential utility of rNS3ΔHD fusion protein as candidate diagnostic reagent in developing an indirect-ELISA for sero-surveillance of animals for BTV antibodies under DIVA strategy, wherever monovalent/polyvalent killed BT vaccine formulations devoid of NS proteins are being practiced for immunization.

A coiled-coil motif in non-structural protein 3 (NS3) of bluetongue virus forms an oligomer.

Bluetongue, an arthropod-borne non-contagious hemorrhagic disease of small ruminants, is caused by bluetongue virus (BTV). Several structural and non-structural proteins encoded by BTV have been associated with virulence mechanisms. In the present study, the NS3 protein sequences of bluetongue viral serotypes were analyzed for the presence of heptad regions and oligomer formation. Bioinformatic analysis of NS3 sequences of all 26 BTV serotypes revealed the presence of at least three coiled-coil motifs (CCMs). A conserved α-helical heptad sequence was identified at 14-26 aa (CCM-I), 185-198aa (CCM-II), and 94-116 aa (CCM-III). Among these, CCM-I occurs close to the N-terminus of NS3 and was presumed to be involved in oligomerization. Furthermore, the N-terminus of NS3 (1M-R117 aa) was over-expressed as a recombinant fusion protein in a prokaryotic expression system. Biochemical characterization of recombinant NS3Nt protein revealed that it forms SDS-resistant dimers and high-order oligomers (hexamer and/or octamer) under reducing or non-reducing conditions. Coiled-coil motifs are believed to be critical for NS protein oligomerization and have potential roles in the formation of viroporin ring/pore either with six/eight subunits and this is the first study toward characterization of CCMs in NS3 of bluetongue virus.

Cloning and sequence analysis of a partial CDS of leptospiral ligA gene in pET-32a - Escherichia coli DH5α system.

AIM: This study aims at cloning, sequencing, and phylogenetic analysis of a partial CDS of ligA gene in pET-32a - Escherichia coli DH5α system, with the objective of identifying the conserved nature of the ligA gene in the genus Leptospira. MATERIALS AND METHODS: A partial CDS (nucleotide 1873 to nucleotide 3363) of the ligA gene was amplified from genomic DNA of Leptospira interrogans serovar Canicola by polymerase chain reaction (PCR). The PCR-amplified DNA was cloned into pET-32a vector and transformed into competent E. coli DH5α bacterial cells. The partial ligA gene insert was sequenced and the nucleotide sequences obtained were aligned with the published ligA gene sequences of other Leptospira serovars, using nucleotide BLAST, NCBI. Phylogenetic analysis of the gene sequence was done by maximum likelihood method using Mega 6.06 software. RESULTS: The PCR could amplify the 1491 nucleotide sequence spanning from nucleotide 1873 to nucleotide 3363 of the ligA gene and the partial ligA gene could be successfully cloned in E. coli DH5α cells. The nucleotide sequence when analyzed for homology with the reported gene sequences of other Leptospira serovars was found to have 100% homology to the 1910 bp to 3320 bp sequence of ligA gene of L. interrogans strain Kito serogroup Canicola. The predicted protein consisted of 470 aminoacids. Phylogenetic analysis revealed that the ligA gene was conserved in L.interrogans species. CONCLUSION: The partial ligA gene could be successfully cloned and sequenced from E. coli DH5α cells. The sequence showed 100% homology to the published ligA gene sequences. The phylogenetic analysis revealed the conserved nature of the ligA gene. Further studies on the expression and immunogenicity of the partial LigA protein need to be carried out to determine its competence as a subunit vaccine candidate.

Homogeneity of VacJ outer membrane lipoproteins among Pasteurella multocida strains and heterogeneity among members of Pasteurellaceae.

Outer membrane lipoproteins are widely distributed in Gram-negative bacteria which are involved in diverse mechanisms of physiology/pathogenesis. Various pathogenic bacterial strains belonging to the family-Pasteurellaceae have several surface exposed virulence factors including VacJ/VacJ-like lipoproteins. In the present study, vacJ gene encoding for VacJ outer membrane lipoprotein of different Pasteurella multocida strains (n = 10) were amplified, sequenced and compared with available VacJ/VacJ-like sequences (n = 45) of Pasteurellaceae members. Comparative multiple sequence analysis at amino acid level indicated absolute homogeneity of VacJ lipoprotein among different P. multocida strains. However, heterogeneity (18.0-89.9%) of VacJ lipoprotein was noticed among members of Pasteurellaceae. A predicted lipobox motif (L-3-[A/S/T/V]-2-[G/A]-1-C) was found to be conserved between 12-32aa residues at N-terminus among all VacJ sequences. Bioinformatic analysis indicated that VacJ is a chromosomal gene product exposed on the bacterial surface, possibly essential for either physiological or pathogenicity process of Pasteurellae and distributed widely among P. multocida serogroups. The study indicated potential possibilities of using absolutely conserved VacJ lipoprotein either as 'signature gene/protein' in developing diagnostic assay or as a recombinant subunit vaccine for P. multocida infections in livestock.