Construction Of PcDNA3.1(+)/ICP27 Recombinant DNA Vaccine Against HSV-2 And Its Expression In Eukaryotic Cells

Objective: To construct a new DNA vaccine against Herpes Simplex Virus type II and identify its expression in eukaryotic cells for the following research of immunogenicity of the vaccine .Methods: Culture and amplificate HSV-2 virus in cultured vero cell, isolate total RNA, amplificate UL54 gene(expressing protein ICP27) of full-length by RT-PCR, and then connect UL54 gene to the pMD19-T Simple Vector and transform into E. coli DH5α, extract plasmids. Make UL54 gene and eukaryotic expression plasmid—pcDNA3.1 (+) containing cohesive ends by KpnI and XbaI Double restriction enzyme digestion. Construct recombinant plasmid—pcDNA3.1 (+) / ICP27 (viz. HSV-2 DNA vaccine), and then identificate if success by double restriction enzyme digestion and sequencing. Transfect the recombinant plasmid into COS-7 cells by method of liposome-mediated transfection, identificate the transcription of HSV-2 DNA vaccine in cos-7 cells by RT-PCR and expression by Western blotting.Results: We successfully constructed pcDNA3.1 (+) / ICP27 plasmid: a 5428bp band (the pCDNA3.1 (+) vector) and a 1551bp (HSV-2 UL54 sequence) band were shown by double restriction enzyme digestion; the DNA sequencing of UL54 was compatible with UL54 sequence in Genbank .After transfection into COS-7 cell, a length of 184bp UL54-specific fragments was successfully detected by RT-PCR, approximately 53kDa ICP27 was detected by Western blotting.Conclusion: We successfully constructed a new type of DNA vaccine against herpes simplex virus type II and then made it expressed in eukaryotic cells, so we constructed a solid foundation for further study of immunogenicity of the vaccine.