Journal of Immunology and Immunopathology

Total RNA of IBD (Infectious Bursal Disease) virus strain S394 propagated in chick embryo fibroblast cell culture was isolated. Reverse transcription was performed on total RNA using M-MLV reverse transcriptase, specific primers and polymerase chain reaction (PCR) was done using complementary DNA as template and Taq DNA polymerase as enzyme. Different cyclic conditions were used for different PCR and the product was analyzed on 0.7% or 1.5% agarose gel. The PCR product was confirmed for VP2 gene by nested and seminested PCR and restriction enzyme digestion. RE digestion of PCR product using restriction enzymes BamHI, AccI and EcoRI yielded expected sized DNA bands confirming the VP2 gene. The VP2 gene was cloned into pDrive cloning vector (having ‘3′ U overhang) (Qiagen) and pTargeT (Promega) mammalian expression vector having ‘T’ overhangs. The recombinant plasmid thus obtained was designated as pDrive.ibdvp2 and pTargeT.ibdvp2 respectively. Restriction enzyme analysis of recombinant plasmid pDrive.ibdvp2 was done with enzyme EcoRI, BamHI, AccI and DraI while restriction enzyme analysis of pTargeT.ibdvp2 was done with enzymes EcoRI, BamHI, HindIII, AccI, SacI, ApaI and SalI. On running the PCR product and digested products on 0.7% agarose gel, expected sized fragments were obtained thus confirming the VP2 gene in right orientation in the recombinant plasmids pDrive.ibdvp2 and pTargeT.ibdvp2. Sequencing of the recombinant plasmid was done and VP2 gene sequence was submitted to GenBank and assigned accession no. AJ621158. The VP2 sequence was then analyzed with DNASTAR software and The VP2 protein sequence was also submitted to Swiss-Prot and assigned the accession no. P61825. Molecular modeling of the VP2 gene was done using DNASTAR software.