AI Article Synopsis
- An infectious cDNA clone of the H120 vaccine strain of infectious bronchitis virus (IBV) was successfully created to explore its use as a gene transfer vector.
- Researchers designed primers to amplify and sequence the entire genome of the H120 strain, replacing a specific open reading frame with an enhanced green fluorescent protein (EGFP) gene.
- The resulting recombinant virus was confirmed and studied for its characteristics, indicating that the ORF5a region is a suitable target for inserting recombinant genes in future IBV gene transfer applications.
Article Abstract
An infectious cDNA clone of H120 vaccine strain of infectious bronchitis virus (IBV) was constructed to demonstrate its potential as a gene transfer vector. Primers were designed according to the published genome sequence of H120 strain, and ten cDNA fragments covering the entire genome of H120 strain was amplified by RT-PCR. All the PCR products were ligated into pMD19-T vector and sequenced, and the ORF5a open reading frame in the pMDTM9 plasmid was replaced by an enhanced green fluorescent protein (EGFP) gene. Recombinant plasmids were digested by the restriction enzyme Bsa I, and all the cDNA fragments were recovered. By using appropriate ligation strategy, the genomic cDNA of H120 strain were reconstituted. Then genome RNA was synthesized in vitro by T7 RNA polymerase and transfected into BHK-21 cells. Recombinant virus expressing the green fluorescent protein was rescued and identified by RT-PCR and sequencing. The characteristics of recombinant virus were evaluated by passage in embryonated chicken eggs. This study showed that the 5a ORF is a good candidate for an insertion site of recombinant genes for the development of IBV infectious cDNA clone as a gene transfer vector.
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