Preparation of a dendritic cell vaccine modified by the adenovirus-mediated HSV-2 gD gene
- VernacularTitle:腺病毒介导 HSV-2 gD 基因修饰的树突状细胞疫苗制备
- Author:
Jianyong FAN
;
Ying WANG
;
Huilan YANG
;
Jie LIANG
;
Cuihua LI
- Publication Type:Journal Article
- Keywords:
Herpes simplex virus;
Dendritic cell;
Vaccine
- From:
Journal of Medical Postgraduates
2015;(1):20-24
- CountryChina
- Language:Chinese
-
Abstract:
Objective Up to now, there has been no sure cure for genital herpes (GH), and vaccine seems a most promis-ing approach to the prevention and treatment of herpes simplex virus Ⅱ(HSV-2) infection.In this study, we investigated the feasibili-ty of preparing a dendritic cell ( DC) vaccine modified by the adenovirus-mediated HSV-2 gD gene. Methods We subcloned the HSV-2 gD gene into the vector Shuttle-2 and constructed the recombinant adenovirus pAdeno-HSV-2 gD following identification by en-zyme digestion and DNA sequence analysis .We isolated DCs from the mouse bone marrow , analyzed their phenotypes by flow cytome-try after transfection with the recombinant adenovirus pAdeno-HSV-2 gD, and determined the expression of HSV-2 gD by immunohisto-chemistry, RT-PCR, SDS-PAGE, and Western blot. Results Based on HSV-2 DNA, the corresponding target fragments were am-plified with the gD gene primers.Agarose gel electrophoresis showed the correct size of the PCR product (1182 bp) as predicted.The recombinant adenovirus pAdeno-HSV-2 gD was obtained by transfecting the 293 cells with pAdeno-gD DNA, which had an activity of 4 ×1010 IU/mL.The contents of CD40, CD80, and CD86 were (74.2 ±3.9), (73.9 ±4.1), and (76.1 ±5.5) % in the mature DCs and (81.3 ±3.1), (80.4 ±2.9), and (83.7 ±3.9) % in the pAdeno-HSV-2 gD DCs, significantly increased as compared with those in the immature DCs ([9.7 ±0.5], [7.5 ±1.2], and [5.2 ±1.1] %) (P<0.01).No statistically significant differ-ences were found between the expression of the surface molecules in the pAdeno -HSV-2 gD DCs and that in the cytokine stimulation-in-duced mature DCs (P>0.05).RT-PCR and immunohistochemistry confirmed the expression of HSV-2 gD in DCs.SDS-PAGE and Western blot of the expressed protein showed a new band with an apparent molecular mass corresponding to the predicted size (43000). Conclusion The results of our study have paved the ground for the successful preparation and identification of a dendritic cell vaccine modified by the adenovirus-mediated HSV-2 gD gene.
