Construction and expression of eukaryotic expression vector pcDNA3-ICOSIg / 中国组织工程研究

ABSTRACT

BACKGROUND:So far, inducible co-stimulator is the important costimulatory molecule family member. Inducible co-stimulator can promote the activation of T cel s proliferation and secretion, regulate Th1/Th2 cel polarization dependence, enhance B cel function which depend on the T cel s. So, blocking the inducible co-stimulator may result the inactivation and no reaction of cloning in T cel s, thus inducing the immune escape of tumor on the body. OBJECTIVE:To build a plasmid expression of inducible co-stimulator Ig, in order to observe the expression in rat body. METHODS:cDNA encoding the extracel ular domain of human inducible co-stimulator was prepared. The encode of the domain was fused with the gene of immunoglobulin IgG constant fragment (Ig) of encoding mouse, in order to build the inducible co-stimulator Ig fusion gene and the secreted eukaryotic expression vector pcDNA3-inducible co-stimulator Ig. Enzyme digestion of the recombinant and sequencing was performed, and then the positive liposome coated pcDNA3-inducible co-stimulator Ig was transferred into the muscle tissue of mouse right thigh. Western blot was used to detect the level of inducible co-stimulator Ig. RESULTS AND CONCLUSION:The sequencing confirmed that the size of target gene fragment pcDNA3-inducible co-stimulator Ig plasmid was exactly the same with the sequence of inducible co-stimulator published on Genebank, which indicated the successful of plasmid construction. After transferred into the mouse for 7 days, the liposome coated pcDNA3-inducible co-stimulator Ig was positively expressed in the mice serum, which showed that pcDNA3-inducible co-stimulator Ig could be expressed in the rat muscle cel s. The results suggest that gene synthesis and recombinant technology can successful y construct the eukaryotic expression vector pcDNA3-inducible co-stimulator Ig.