[Liver tissue-specific stable expression of human CD81 molecule].
OBJECTIVE
To express stably human CD81 gene on mouse hepatoma cell line Hepa 1-6 using liver specific promoter.


METHODS
RNA were isolated from human HepG2 cells which could be infected with hepatitis C virus. RT-PCR was carried out using human CD81 gene specific primers. Amplified fragments were cloned into pGEM-T vector. Albumin promoter and enhancer which were liver tissue specific were ligated to the 5'end of human CD81 gene and SV40 polyA sequence was fused with 3'end of CD81. The fused CD81 gene was inserted into eukaryotic expression vector pcDNA3 to construct a recombinant vector pcDNA3-Alb p-CD81 which was then transfected into Hepa 1-6 cells through lipofectamine mediation. Human CD81 mRNA transcription and its protein expression were detected by RT-PCR and FACS, respectively.


RESULTS
Sequence analysis showed that the cloned gene segment was human CD81 gene sequence. After transfection, transcripted human CD81 mRNA was obtained and human CD81 molecules were expressed stably on Hepa 1-6 cells.


CONCLUSIONS
The obtained positive cell clones which stably express HCV receptor human CD81 lay the foundation for further study on interactions between HCV envelope proteins and human CD81, screening of HCV infection blocking drugs and development of HCV infection mouse model.
Jia
Shuai-zheng
SZ
Institute of Transfusion Medicine, Beijing 100850, China. jiasz@nic.bmi.ac.cn
Lu
Li-ping
LP
Liu
Min-xia
MX
Zhan
Lin-sheng
LS
Wang
Hai-ping
HP
Wang
Quan-li
QL
D000818:Animals; D015703:Antigens, CD / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D004195:Disease Models, Animal; D015870:Gene Expression; D006526:Hepatitis C / Q000209:etiology; D006801:Humans; D008099:Liver / Q000378:metabolism*; D051379:Mice; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D060225:Tetraspanin 28
D015703:Antigens, CD; C554798:CD81 protein, human; C554799:Cd81 protein, mouse; D018076:DNA, Complementary; D012333:RNA, Messenger; D060225:Tetraspanin 28