[Construction and expression characteristics in vitro of human papillomavirus type 16 E7 suicidal DNA vaccine].
OBJECTIVE
To construct the suicidal DNA vaccine of human papillomavirus type 16 E7 gene (HPV16), and explore the DNA vaccine expression characteristics in vitro and capacity of inducing the transfected cells into apoptosis.


METHODS
HPV16 E7 gene cloned by PCR from pET32/E7 was inserted into the plasmid pSCA1 to construct the recombinant plasmid pSCA/E7, followed by identification with PCR, BamH I and Sma I digestion and sequencing. pSCA/E7 was then used to transfect BHK-21 cell line. The transient expression of HPV16 E7 gene was confirmed by immuno-fluorescent staining, and the apoptosis induced by pSCA/E7 was checked with TDT-mediated dUTP nick end-labeling (TUNEL).


RESULTS
The cloned E7 gene fragment was about 400 bp in length. PCR, restriction endonuclease digestion and sequence analysis revealed that the HPV16 E7 gene was cloned into the eukaryotic expression plasmid pSCA1 successfully. Immunofluorescent staining confirmed that the E7 gene could express in BHK-21 cell line. The BHK-21 cells transfected with pSCA/E7 could be induced into apoptosis which was confirmed by TUNEL.


CONCLUSIONS
The results show that HPV16 E7 suicidal DNA vaccine can express in BHK-21 cell line, and induce the pSCA/E7 transfected cells into apoptosis. These findings may provide the foundation for exploring the therapeutic vaccine against HPV16-associated cervical cancer.
Wang
Hong-Ren
HR
Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China.
Wang
Bao-Ning
BN
Li
Wan-Yi
WY
Zuo
Feng-Qiong
FQ
Li
Hong
H
Jiang
Zhong-Hua
ZH
Shi
Qiao-Fa
QF
Li
Ming-Yuan
MY
D000818:Animals; D017209:Apoptosis / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D005260:Female; D041722:Genes, Transgenic, Suicide / Q000235:genetics; D006801:Humans; D020287:In Situ Nick-End Labeling; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics / Q000276:immunology*; D050725:Papillomavirus E7 Proteins; D053918:Papillomavirus Vaccines / Q000235:genetics / Q000276:immunology*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D014162:Transfection; D002583:Uterine Cervical Neoplasms / Q000276:immunology / Q000517:prevention & control / Q000821:virology; D019444:Vaccines, DNA / Q000235:genetics / Q000276:immunology*
D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D053918:Papillomavirus Vaccines; D011994:Recombinant Proteins; D019444:Vaccines, DNA; C059731:oncogene protein E7, Human papillomavirus type 16