[Construction and identification of a recombinant lentivirus vector of latent membrane protein 1].
OBJECTIVE
To construct a recombinant lentivirus vector of latent membrane protein 1 (LMP1) and detect the expression of LMP1 in vitro.


METHODS
The LMP1 fragment including all the exons was amplified by PCR and inserted to the downstream of CMV promoter in the lentivirus vector pCDF. The three plasmids (packaging plasmid pFIV-34N, envelope plasmid pVSV-G and target plasmid pCDF-LMP1) were packaged into 293FT cells via liposome. The virus supernatant was harvested, concentrated and titrated. Mouse B lymphoma cell line A20 was transfected with the recombinant lentivirus vector of LMP1, and the expression of LMP1 in A20 cells was detected by RT-PCR and Western blotting.


RESULTS
DNA sequencing confirmed that the sequence of PCR-amplified LMP1 was consistent with the GenBank data. The LMP1 gene fragment was cloned into pCDF in the right direction, and the open reading frame of LMP1 was maintained. The 3 plasmids were effectively transferred into 293FT cells, which emitted green fluorescence in the cytoplasm and on the cell membrane under fluorescence microscope. The titer of the lentivirus vector reached 10(7) Tu/ml with a transfection efficiency 90% in A20 cells. LMP1 expression was detected by RT-PCR and Western blotting in transfected A20 cells.


CONCLUSIONS
The recombinant lentivirus vector of LMP1 constructed can be effectively transfected into A20 cells, which provides a basis for exploring the role of LMP1 in the pathogenesis of lymphoma.
Chen
Xiao-yan
XY
Department of Pathology, Nanfang Hospital, Cancer Research Institute, Southern Medical University, Guangzhou 510515, China. dcxy128@163.com
Zhang
Gong
G
Liu
Fang
F
Li
Hui-ling
HL
Fang
Wei-yi
WY
Jiang
Qing-ping
QP
Zhao
Tong
T
D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D016393:Lymphoma, B-Cell / Q000473:pathology; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics*
C037662:EBV-associated membrane antigen, Epstein-Barr virus; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D049452:Green Fluorescent Proteins