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Expression and functional analysis of murine intercellular adhesion molecule 1 (ICAM-1)

University of British Columbia

Cell adhesion molecules enhance Interactions between adjacent cells In order to mediate a large variety of functions of the Immune system. An antibody against the murine lymphocyte surface antigen MALA-2 has previously been shown to Inhibit mixed lymphocyte response. A λgt10 cDNA library from NS-1 cells was screened and a cDNA clone, K3-1.1, was previously isolated. It had significant homology to the human ICAM-1 gene. This thesis covers the isolation of a second cDNA clone, K4-1.1, and its comparison to K3-1.1 In terms of expression, function and distribution. The two clones are identical in sequence with the exception of the 5’ ends. Expression of these two clones was examined using a transient expression system of COS cell transfection. Cell surface expression of the K3-1.1 clone could not be detected by FACS analysis. Even when the 5' untranslated region of the K3-1.1 clone (which has 10 potential translation start sites) was removed, protein could not be detected at the cell surface, intracellularly, or extracellularly. However, K4-1.1 expression was detected at the cell surface. Northern blot analysis reveals that there are two distinct messages which are likely to be represented by the two clones. When the northern blot was probed with the 5' end of the K3-1.1 clone, only one of the messages was detected. This together with the result of Southern blot analysis suggests that the two messages are likely the result of alternate splicing. In order to examine the interactions of the murine ICAM-1 with the surface of other cells, an expression system which would produce large amounts of a secreted soluble form was established. The soluble protein was purified from the supernatant of transfected cells by an antibody-affinity column and used in preliminary binding assays.

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2010-10-05

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http://hdl.handle.net/2429/28939

Medical Genetics

University of British Columbia

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