[Construction of hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 and identification of its expression].
OBJECTIVE
To construct hepatoma-targeting recombinant co-expression adenovirus vector of Staphylococcal enterotoxin A (SEA) and CD80 gene.


METHODS
Us-ing the adenovirus transfer plasmids pShuttle and pShuttle-CMV, we constructed a new transfer plasmid pShuttle2 with polyA signal sequence instead of CMV enhancer/promoter. AFP enhancer, promoter, SEA or CD80 gene was subcloned into pShuttle2 from the vectors pKS-EP or pMD18-T-BIS respectively, and then the constructed plasmid pShuttle2-BIS containing AFP enhancer, promoter, SEA or CD80 gene was cotransformed into E.coli BJ5183 with backbone vector pAdEasy-1 to obtain recombinant adenovirus DNA. The recombinant adenovirus DNA was transfected into 293 cells to prepare adenovirus. After AFP-producing cell line Hepa1-6 and AFP-nonproducing cell lines B16 and NIH3T3 were infected by recombinant adenovirus, the expression of SEA and CD80 on the surface of cells was detected by indirect immunofluorescent staining, laser confocal microscope and flow cytometry (FCM).


RESULTS
SEA and CD80 was specifically co-expressed on the surface of infected Hepa1-6 cells but not on B16 and NIH3T3 cells.


CONCLUSIONS
Hepatoma-targeting recombinant co-expression adenovirus vector of SEA and CD80 gene was successfully constructed, which lays the foundation for further research on application of SEA and CD80 in targeted gene therapy for hepatoma and the underlying immunological mechanisms.
Si
Shao-yan
SY
Department of Pathology, Fourth Military Medical University, Xi'an 710032, China. sishy306@yahoo.com.cn
Sui
Yan-fang
YF
Hu
Pei-zhen
PZ
Zhang
Xiu-min
XM
Ge
Wei
W
Li
Zeng-shan
ZS
Huang
Yang
Y
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D018122:B7-1 Antigen / Q000235:genetics*; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000378:metabolism / Q000473:pathology / Q000628:therapy; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004274:DNA, Recombinant / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D004768:Enterotoxins / Q000235:genetics*; D005260:Female; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D015870:Gene Expression; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013210:Staphylococcus
D018122:B7-1 Antigen; D004274:DNA, Recombinant; D004279:DNA, Viral; D004768:Enterotoxins; C026066:enterotoxin A, Staphylococcal; D004262:DNA Restriction Enzymes