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KMID : 0357119930150020129
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Korean Journal of Immunology
1993 Volume.15 No. 2 p.129 ~ p.137
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Expression of Human CTA-4 Extracellular Domain in Escherichia coli
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Park Chang-Hwan
Seo Seung-Hyun
Chung Yong-Hoon
Cho Yang-Ja
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Abstract
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CTLA-4(cytotoxic T lymphocyte associated antigena-4), together with CTLA-1, CTLA-2 and CTLA-3, was identified by reverse genetic approach employing substrative hybridization followed by a series of differential hypbridizations. Among these 4 novel genes associated with T cell activation only CTLA-4 is known to exhibit a typical membrane protein structure. And this molecule is homologous to CD28, an accessory molecule providing an important signal for T cell activation, with 128% of amino acid and 67% of nucleic acid identity. Contrary to CD28 the functional role of CTLA-4 in T cell activation remains unaswered because the natural protein product of this molecule has not yet been described. This study was undertaken to make recombinant protein antigen for raising antibody specific to human CTLA-4 molecule. Human CTLA-4 cDNA was amplified from phytohemagglutinin-stimulated human peripheral blood lymphocytes mRNA by using reversc transcription-polymerase chain reaction(RT-PCR). In this reaction the optimal annealing temperature was 52℃and about 370 base paired ds DNA band was amplified. This amplified human CTLA-4 DNA was subsequently cloned into pET22b vector and sequenced by using dideoxy chain termination method. The sequence was identical to the sequence reported previously except the 331st base of protein coding region was changed from guanylic acid to adenylic acid. Cloned plasmid was introduced into HMS174(λDE30 competent cell and the human CTLA-4 extracellular domain fusion protein was induced by adding IPTC. The molecular weight of this protein was about 18kD. Regardless of the problems of protein folding in prokaryocyte and fused foredign peptide sequence this overexpressed protein would be useful antigen, at least for raising anti-human CTLA-4 polyclonal antibody.
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KEYWORD
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CTLA-4, Protein expression
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