Construction of Recombinant Adenoviruses Carrying Rotavirus Gene
Date Issued
2005
Date
2005
Author(s)
MA, PEI-RU
DOI
zh-TW
Abstract
Abstract
Rotaviruses, which form one genus of the family Reoviridae, are now recognized as the most important cause of severe viral gastroenteritis in infant and pre-school children. Because of the widespread nature of rotavirus disease, a safe and effective rotavirus vaccine is urgently needed, particularly in developing countries. The currently available live, attenuated oral rotavirus
vaccine is promising, but improved vaccines are still needed.
Recombinant adenovirus (rADV) vectors have received considerable attention for gene therapy because of their high transduction efficiency, infection of both dividing and resting cells of many cell types, including antigen-presenting cells, a feature that has called much attention upon them both as vaccine delivery vehicles.
This study reports construction of rADV carrying a single gene, either VP7 or VP4 gene, and rADV carrying both VP6 and NSP4 genes of G9P[8] rotavirus. First, the plasmids carrying rotavirus gene, pShuttle-CMV/VP4, pShuttle- CMV/VP7 and pShuttle/VP6-NSP4, were constructed and co-transformed with adenovirus gene ( pAdEasy-1 ) into the E. coli BJ5183. After homologous recombination, the plasmid was purified and transfected into 293A cell line which could provide E1 protein that is necessary for rADV replication.
The expression of rADV ( rADV/VP4-Mu, rADV/VP7 and rADV/VP6-NSP4 ) carrying the target gene was evaluated by polymerase chain reaction ( PCR ), western blotting and Immuno-fluorescence staining ( IFA ). rADV carrying only pShuttle-CMV（rADV/control）was used as the control.
For confirmation of rADV carrying rotavirus gene after replication in 293A cell, DNA extracted from the infected cell was subjected to PCR. At present, the expression of the target protein could not be detected by western blotting. More experiments should be done to see if the target protein had been expressed in 293A cell and what made the differences in expression between adenovirus protein and target protein。
Subjects
輪狀病毒
VP7
VP4
VP6
NSP4
重組腺病毒
Rotavirus
Recombinant adenovirus
SDGs
Type
other
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