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KMID : 0357319940290020205
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Journal of the Korean Society for Microbiology
1994 Volume.29 No. 2 p.205 ~ p.216
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Cloning and Expression of Human CTLA-4 cDNA in Murine L Cell
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정용훈
장경덕/박장환/김정목/조양자
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Abstract
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When T cell activated a number of new antigens are induced transiently on it's surface. 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
origirally
identified by the differential screening following subtractive hybridization of murine cytotoxic T cell cDNA library and mapped to the same location on chromosome 2 in human and chromosome 1 in mouse where CD28 located. 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-polymerase chain reaction (RT-PCR) method. By using this RT-PCR technique we could amplify target cDNA from source mRNA in a single tube in only 3-4 hours. And amplified human CTLA-4 cDNA was subsequently cloned into pRC/CMV
vector. In double stranded DNA sequencing the 49 th and 331 st base of protein coding region have been changed from adenine to guanine and from guanine to adenine respectively. The changes in these two bases substituted the 17th and 111th amino
acid of
human CTLA-4 from threonine to alanine and 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 the variation of glycosylation.
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KEYWORD
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