[Construction, identification and expression of three kinds of shuttle plasmids of adenovirus expression vector of hepatitis C virus structure gene].
OBJECTIVE
To construct three recombinant shuttle plasmids of adenovirus expression vector which can express hepatitis C virus(HCV) different structure genes(C, C+E1, C+E1+E2) in order to pack adenovirus expression vectors which can express HCV different structure gene effectively.


METHODS
The different HCV structure genes derived from the plasmid pBRTM/HCV1-3011 by using polymerase chain reaction (PCR) were inserted into the backward position of cytomegalovirus(CMV) immediate early promotor element of shuttle plasmid(pAd.CMV-Link.1) of adenovirus expression vector respectively, then the three recombinant plasmids (pAd.HCV-C, pAd.HCV-CE1, pAd.HCV-S) were obtained. The recombinant plasmids were identified by endonuclease, PCR and sequencing. HCV structure genes were expressed transiently with Lipofectamine 2000 coated in HepG2 cells which were confirmed by immunofluorescence and Western-Blot.


RESULTS
Insert DNAs of the three recombinant plasmids' were confirmed to be HCV different structure genes by endonuclease, PCR and sequencing. The three recombinant plasmids can express HCV structure gene (C, C+E1, C+E1+E2) transiently in HepG2 cells which were confirmed by immunofluorescence and Western-Blot.


CONCLUSIONS
The three recombinant shuttle plasmids of adenovirus expression vector can express HCV structure gene(C, C+E1, C+E1+E2) transiently. This should be useful to pack adenovirus expression vector which can express HCV structure genes.
Cao
Yi-zhan
YZ
Department of Emergency Medicine, Affiliated Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, Shanxi, China.
Hao
Chun-qiu
CQ
Feng
Zhi-hua
ZH
Zhou
Yong-xing
YX
Li
Jin-ge
JG
Jia
Zhan-sheng
ZS
Wang
Ping-zhong
PZ
D000256:Adenoviridae / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics*; D015394:Molecular Structure; D010957:Plasmids; D015678:Viral Structural Proteins / Q000235:genetics*
D015678:Viral Structural Proteins