Cloned Vero cell lines transfected with full-length A-segment or ORF1 cDNA sequence of IBDV.
Recombinant plasmids containing the A-segment or VP2/4/3 gene of infectious bursal disease virus (IBDV) were transfected into Vero cells. Monoclonal Vero cell lines were generated under G418 selection. Genomic (PCR/Southern blot) and transcriptional (RT-PCR/Northern blot) analyses showed that one copy of A-segment or VP2/4/3 or a partial gene of them was randomly and stably inserted into genomic DNA of Vero cells, and was able to transcribe corresponding mRNA. IFA/IPMA and Western blot analyses further confirmed that two of the monoclonal Vero cells with insertion of the A-segment of IBDV into genomic DNA could stably express VP2, VP3 and VP5 proteins, one cell line only expressed VP2 protein, and three monoclonal Vero cell lines with genomic insertion of the VP2/4/3 gene of IBDV could express VP2, VP3 and VP4 proteins. Under G418 selection, integrated foreign genes can be inherited along with cellular genomic DNA during cell replications. Moreover, DNA fragmentation and caspase-3 activity assays illustrated that cell apoptosis did not develop in monoclonal Vero cell lines expressing VP2 and VP5 proteins. The monoclonal IBDV gene-inserted Vero cell lines developed in this study will facilitate better understanding of IBDV and other members of the Birnaviridae in an expression system that would enable investigation of virus-host cell interactions on the cellular and molecular level.
Ye
Juxiu
J
Laboratory of Virology and Immunology, Institute of Preventive Veterinary Medicine, College of Animal Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou, Zhejiang 310029, PR China.
Chen
Qingxin
Q
Zhou
Jiyong
J
Li
Lingyan
L
D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015139:Blotting, Southern; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002642:Chick Embryo; D002522:Chlorocebus aethiops; D002999:Clone Cells; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000235:genetics*; D004351:Drug Resistance; D005786:Gene Expression Regulation; D005814:Genes, Viral / Q000235:genetics*; D016678:Genome / Q000235:genetics; D007243:Infectious bursal disease virus / Q000235:genetics*; D009355:Neomycin; D016366:Open Reading Frames / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism
D018076:DNA, Complementary; D012333:RNA, Messenger; D014764:Viral Proteins; D053148:Caspase 3; D009355:Neomycin