Construction of a recombinant Bak1 -expressed adenovirus vector and its effect on human osteosarcoma cells line
CHAI Shuang
HUANG Hong
WAN Lei
WANG Ji-li
WANG Wei
HUANG Hong-xing
Abstract：Objective To construct a recombinant BCL2-antagonist/killer 1 (Bak1)-expressed adenovirus , thus to establish a cell model by infecting human osteosarcoma MG 63 cells, and to study the effect of recombinant Bak 1 adenovirus on MG63 cells.Methods The full-length cDNA of Bak1 was prepared through qPCR .Plasmid pENTER-Bak1 containing Bak1 gene was constructed by using adenovirus shuttle plasmid pENTER , and the sequence was identified by restriction enzyme digestion method .After linearized by Enzyme digestion of Pme I , it was recombined with BJ5183 cells which contained adenoviral back -bone plasmid pAd -Amp.The recombinant adenovirus vector was linearized by Pac I enzyme and transfected into HEK -293 cell.Bak1 recombinant adenovirus was extracted by repeated centrifugation and low temperature cracking .Real -time PCR was applied to measure the adenovirus titer .MG63 cells were infected with recombinant adenovirus .The expression of Bak1 was assessed with qPCR and Western blot .The cell activity was de-termined by MTT.Results It was proved by gene sequencing and enzyme digestion that pENTER -Bak1 was successful-ly constructed.The expression of Bak1 gene was confirmed by qPCR , and the expression of Bak1 protein was confirmed by Western blot.MTT showed that the recombinant expression of Bak 1 adenovirus could weaken the viability of MG 63 cells. Conclusion Bak1 recombinant adenovirus vector is successfully constructed , and the recombinant adenovirus vector could infect MG63 cells and express Bak1 protein.Bak1 adenovirus could weaken the viability of MG 63 cells.
Keywords：BCL2-antagonist/killer 1(Bak1)recombinant adenovirus vectorhuman osteosarcoma cells
Publication Date：2018-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 1332-1336 )