Overexpressed Human TNF
Receptor-75 Can Independently Mediate hTNFα-induced Cytotoxicity in BHK-21
Cells
FANG Jia, WANG De-Bao1 and CHEN Chang-qing
( Shanghai Research Center of Biotechnology, the Chinese Academy of
Sciences, Shanghai 200233, China； 1State Key Laboratory of Molecular Biology, Shanghai Institute of
Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China )
DUCH Mogens Ryttergaard and PEDERSEN Finn Skou
( Department of Molecular and Structural Biology, Aarhus University, Aarhus
DK 8000, Denmark )
Abstract A
recombinant bicistronic expression vector was constructed containing human TNF
receptor-75 gene and encephalomyocarditis virus(EMCV)internal ribosome entry
site(IRES), followed by a neomycin phosphotransferase gene as selectable
marker. BHK-21 cells were transfected with this vector using electroporation
method and positive clones overexpressing the genes of interest were obtained
after selection with G-418. The expression of two hTNFRs in those cells at RNA
transcriptional and the protein translation levels had been proved by RT-PCR
and indirect ELISA. It was found that the hTR75 could mediate cytotoxicity
independently in BHK-21 cells after those cells were treated by hTNFα.
Furthermore, hTR55 did not display the synergistic activity on this function of
hTR75. These results put forward a new way for further studies on structure and
function relationships of hTR75 and the interactions between two hTNFRs.
Key Words hTNF receptors； expression； internal ribosome entry site； signal transduction
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