Identification of CTCL tumor antigens by SEREX / 解放军医学杂志

ABSTRACT

Objective To isolate and purify the membrane protein of SeAx cells, which was derived from cutaneous T-cell lymphoma cell line (CTCL), and to prepare polyclonal antibodies against membrane proteins. CTCL antigens were screened with SEREX method from the cDNA expression library of SeAx cells. Methods SeAx cells were cultured to 2?1010 and membrane proteins were purified by means of differential centrifugation after homogenate was made, and organelle protein specific antibodies were used to perform Western blot. Rabbit was immunized 4 times by the membrane proteins, and the serum antibody titer was measured by ELISA after each immunization. The specific interactions between antibodies in serum and SeAx proteins were detected by Western blotting. E. coli were initially transfected by recombinant phages, and the recombinant proteins expressed were transferred onto the nitrocellulose membranes, which were then again reacted with diluted serum from the immunized rabbit. Positive clones were subcloned and the nucleotide sequence of the cDNA inserts was determined. Finally, cDNA sequence analysis via database searching was performed to define the gene. Results Membrane proteins of SeAx cells were purified by differential centrifugation and a 1?10-5 titer of antibody was obtained after 4 times of immunization. 1?106 phage clones were screened, 25 of which were positive. Nucleotide sequencing and database searching showed that 4 cDNA inserts were cell membrane proteins, respectively as integrin alpha 4,ligatin, matrix metalloproteinase 24 and MHC-I molecule HLA-A. Conclusion Membrane proteins of CTCL and SeAx cells, are purified and polyclonal antibodies against SeAx cell membrane proteins are successfully prepared. The 4 genes of membrane proteins are identified by means of SEREX from SeAx cDNA library, expressed by phages. These genic candidates are able to induce systemic humoral immune reactions and might therefore be promising targets for immunotherapeutic interventions of CTCL.