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KMID : 0382619930130020587
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Hanyang Journal of Medicine
1993 Volume.13 No. 2 p.587 ~ p.598
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Cloning of Human CTL-4 cDNA by Using Reverse Transcription-polymerase Chain Reaction and It's Expression in Murine L Cell
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장경덕
정용훈/박장환/김경희/조양자
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Abstract
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Occupancy of the T-cell receptor (TCR) by antigen in association with class II MHC molecule is required for the initiation of T cell activation. However TCR stimulation is not a sufficient signal to account for all the observed events that occur
during
T cell activation. And in the absence of a second costimulatory signal provided by antigen presenting cell antigen specific signal may lead to clonal inactivation or anergy. CD28, a T cell surface molecule first defined by monoclonal antibody 9.3
on
human cells, appears to function as an alternative, TCR/CD3-independent, activation pathway for T cell. This molecule provides costimulatory signal for T cell proliferation by increasing a number of lymphokines and cytokines production. A T cell
activation antigen CTLA-4 is homologous to CD28 with 28% amino acid and 67% of nucleic acid homology in the protein coding region. CTLA-4 was originally identified by the differential screening following subtractive hybridization of murine
cytotoxic t
cell cDNA library an mapped to the same location on chromosome 2 in human and chromosome 1 in mouse as CD28. These findings and activation associated expression pattern of CTLA-4 raise question about functional role of this molecule in T cell
activation. In this study human CTLA-4 cDNA was amplified from phytohemagglutinin (PHA)-stimulated human peropheral blood lymphocytes mRNA by using reverse transcription-polymearese chain reaction (RT-PCR) method. And amplified human CTLA-4 was
subsequently cloned into pRC/CMV vector. In double stranded DNA sequencing the 331st base of protein codig region was changed from guanylic acid to adentlic acid and this substituted the 111th amino acid of human CTLA-4 from alanine to threonine.
Murine
L cell transfected with this clone expressed 36kD protein. It appears that this difference of molecular weight between native ( 34kD) and recombinant (36kD) human CTLA-4 molecule may due to glycosylation differences. The cDNA and murine L cell
line
expressing human CTLA-4 developed by this study would contribute in future work unveiling the biological role of CTLA-4 in T cell activation.
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KEYWORD
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