[Construction of herpes simplex virus type I glycoprotein D DNA vaccine and its preliminary study].
OBJECTIVE
The goal of this study was to construct a eukaryotic expression plasmid containing the gene encoding herpes simplex virus type I glycoprotein D (HSV-1, gD) and evaluate its utility for DNA immunization in mice.


METHODS
The gD gene was amplified from viral DNA using PCR with EcoR I and BamH I restriction sites encoded on 5' and 3' ends, respectively. The PCR fragment was inserted into the transfer vector pGEM-T Easy. gD was then cut from this vector and inserted into the EcoR I and BamH I sites in the pcDNA3.1 at the multiple cloning sites (MCS). The recombinant plasmid, pcDNA3.1-gD1, was transfected into COS-7 cells using Lipofectamine according to the manufacture's instructions. The expression of the glycoprotein D was analyzed by immunoblotting of the cell lysates. 4-6 weeks old BALB/C mice were given two injections at tibia anterialis muscle, each containing 100 micrograms of plasmid DNA, on days 0 and 15. pcDNA3.1 was used as negative control. Blood samples were taken from all mice at weeks 0, 2, 4, and 6 after the first inoculation. Standard indirect ELISA was employed to evaluate the levels of specific total Ig in serum.


RESULTS
The recombinant plasmid was confirmed with restriction digestion and sequencing to contain target gene segment and expressed in COS-7 cells in vitro shown by Western blotting. The pcDNA3.1-gD1 immunized group induced specific antibody response as compared to the negative control, and the titer was about 1:2000.


CONCLUSIONS
The recombinant plasmid pcDNA3.1-gD1 is potential to be used as a candidate vaccine, for the treatment of HSV-1 infection.
Zhu
Ming-zhao
MZ
Department of Biophysics, Institute of Basic Medical Sciences, CAMS, PUMC, Beijing 100005, China.
Liu
Hong-wei
HW
Liu
Xiao-juan
XJ
Han
Ye-hua
YH
Yang
Bao-ling
BL
Song
Guo-xing
GX
D000818:Animals; D019556:COS Cells / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D019444:Vaccines, DNA / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology
D019444:Vaccines, DNA; D014759:Viral Envelope Proteins; C039447:glycoprotein D, Human herpesvirus 1