To study the expression of herpes simplex virus type 2 latency-associated transcript (LAT) open reading frame 1 (ORF1) and its anti-apoptosis function induced by actinomycin D in Vero cells. The recombinant plasmid pEGFP-ORF1 was constructed and transfected into Vero cells, and the expression of ORF1 was identified by RT-PCR. The changes of Vero cells morphology induced by actinomycin D were observed by fluorescence microscopy, Hochest33258 fluorescence staining. Cells viability was evaluated by MTT assay and cells apoptosis rate was detected by flow cytometry. Double digestion and sequencing confirmed the pEGFP-ORF1 was constructed successfully, RT-PCR showed that the target gene was highly expressed in Vero cells. Hochest33258 staining reaveals that Vero cells transfected with pEGFP-ORF1 and induced apoptosis by actinomycin D had no changes in morphology. MTT assay showed that the viabilities of Vero cells transfected with recombinant plasmid pEGFP-ORF1 and induced apoptosis by actinomycin D has no statistically significant difference compared with the untreated normal control group (P > 0.05), but remarkable higher than Vero cells transfected with empty plasmid pEGFP-C2 and induced apoptosis by actinomycin D, the difference was statistically significant (P < 0.05). Flow cytometry assay shows that the cells apoptosis rate had no significant difference between pEGFP-ORF1 group and the normal group, but the cells apoptosis rate ofpEGFP-ORF1 was lower than the pEGFP-C2 group. HSV-2 LAT ORF1 gene can be expressed in Vero cells and can protect Vero cells from apoptosis induced by actinomycin D.
Animals ; Apoptosis ; physiology ; Cercopithecus aethiops ; Dactinomycin ; Herpes Simplex Virus Protein Vmw65 ; genetics ; Herpesvirus 2, Human ; genetics ; Open Reading Frames ; genetics ; Promoter Regions, Genetic ; Transcription, Genetic ; Vero Cells ; Viral Proteins ; genetics ; Virus Activation ; Virus Latency ; genetics ; physiology