Abstract:

BACKGROUND: The construction of adenovirus vector using Adeasy system is tedious with poor results due to the multiple restriction enzyme digestion, linkage, and screening.
OBJECTIVE: To construct a recombinant adenovirus vector containing LMP-1 gene using Gateway technology.
METHODS: Total RNA was extracted from rat osteoblasts and the LMP-1 gene was acquired by RT-PCR, the LMP-1 gene and entry vector pENTR/D-TOPO were used to create the entry clone with the directional TOPO clone technology, then the entry clone and the expression vector were used to create the expression clone through the LR recombination reaction; at last the adenovirus expression clone was linearized by PacI and transfected to the 293A cell line. The target gene was compared to LMP-1 in the Genebank (Gene number: AF095585), and the expression of pAd-LMP-1 was observed.
RESULTS AND CONCLUSION: The electrophoresis showed that the extracted RNA was integrated, and the purity was accorded with the requirement. LMP-1 gene was successfully acquired. The entry clone was verified by enzymes digestion, and sequencing. The constructed pAd-LMP-1 recombination adenovirus vector was successfully packaged in 293A cells. This result demonstrated that it is easy and rapid to obtain pAd-LMP-1 by constructing pAd-LMP-1 recombination adenovirus vector using the Gateway technology.