Cytotoxic T Lymphocyte-associated Antigen -4 (ctla-4) Gene In Streptomyces And E. Coli Cloning And Expression Study

CTLA-4 (cytotoxic T lymphocyte associated protein-4) is one of members of immunoglobulin superfamily. It is mainly expressed on activated lymphocytes surface which existed as dimer mostly and a few as monomer. The dimer of extracellular domain of CTLA-4 bounding specificity to B7 molecule is 150 folds higher than CD28. CTLA-4 is one of the most important costimulation signal with CD28 in the activation costimulate signal model of T cell and is regarded as the down-regulation factor for the CD28/B7 pathway. The multiple in vivo murine models were demontrating that blocking of this pathway can suppress the autoimmune diseases , allografts rejection and graft verse host disease. It is show that the extracellular domain of CTLA-4 bound specifically to CHO cells transfected with B7 and also immunoprecipitated with B7 on cell surface. The immunosuppressive effects of CTLA-4Ig (a soluble molecule comprising the extracellular domain of CTLA-4 joined to an IgG Cγ1) in vivo proved the therapeutic using for treatment of autoimmune disorder.We obtained the CTLA-4 cDNA by PCR amplification. The pE plasmid which contain hsCTLA-4 cDNA was used as the template. Primers were designed based on the hsCTLA-4 nucletide and matching to the expression plasmid in S.lividans or E.coli, Each PCR products was verified by DNA sequencing.Gene cloning and expression of CTLA-4 was performed in S.lividans. Two different signal peptides-vsi and gpp were used to investigate the secretory expression efficiency in S.lividans. There are two regulation sequence with different length before the vsi promoter, one is 230bp and the other is 10bp. The hsCTLA-4 gene was fused to the two different vsi sequences and verified to be in the apporapriate ORE The fusion genes were inserted into the shuttle vector-pUWL-219 and transformate S.lividans TK24 respectively. The two recombinat strains were named S.lividans[pUWL219-VC] and S.lividans[pUWL219-46] . The hsCTLA-4 was also inserted into the downstream of gpp signal peptide in the plasmid pLNSP. The recombinat strain was named...