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The pGEM-T is 3kb in length, and contains the amplicin resistance gene, conferring selection of the plasmid in E. coli, and the ori site which is the bacterial origin of replication. The plasmid has multiple cloning sites as shown below. The coding sequence was inserted by TA cloning. Many E. coli strains are suitable for the propagation of this vector including JM109, DH5α and TOP10.
The coding sequence can be easily obtained by digesting the vector with proper restriction enzyme(s). The coding sequence can also be amplified by PCR with M13 primers, or primer pair SP6 and T7.
CD52 / CDW52 is a small glycosylphosphatidylinositol (GPI) anchored glycoprotein. It has a mature peptide comprising only 12 amino acids and is abundantly expressed on human lymphocytes. From the clinical point of view this protein is an important target for therapeutic interventions aimed at leukocyte depletion in hematological malignancies and post-transplant immunosuppression. CD52 / CDW52 may play a role in carrying and orienting carbohydrate. It is an unusually good target for complement-mediated cell lysis. CD52 / CDW52 is targeted by alemtuzumab which is an antibody used for the treatment of chronic lymphocytic leukemia and currently being evaluated in a phase III clinical trial for the treatment of multiple sclerosis. Alemtuzumab may be highly effective in large granular lymphocytic leukemia, prospective serial monitoring for the presence of CD52 / CDW52-deficient clonal cytotoxic T-lymphocytes should be a component of clinical trials investigating the efficacy of this drug. CD52 / CDW52 deficiency may explain lack of response to alemtuzumab, and such therapy may confer a survival advantage to glycophosphatidylinositol-negative clonal cytotoxic T-lymphocytes.