Cloning Of VCAM-1 Extracellular Domain D1-D4 CDNA Gene And Its Prokaryotic Or Eukaryotic Expression

VCAM-1 is an important cell adhesion molecule belonging to a member of the immunoglobulin superfamily(IGSF).The molecular weight of VCAM-1 is about 100-110 kD,VCAM-1 extracellular domain proteins have seven domains,D1-D7.Among the extracellular domains,the D1 and D4 domains of VCAM-1 is a specific binding site of very late antigen(VLA-4).It is reported that VCAM-1 involved in many physiological functions,such as regulation of inflammatory reactions,immune response,homing and recirculation of lymphocytes,cell and tissue differentiation,development and participation in tumor proliferation,metastasis.To express a gene encoding VCAM-1 D1-D4,total RNA was extracted from mouse fibroblasts NIH/3T3 cells.After PCR amplification of VCAM-1 D1-D4,the PCR product was subcloned into pMD19-T vector,then cleavaged by double-enzyme digestion from pMD19-T vector and inserted into a pET28a expression vector to construct a recombinant expression plasmid pET28a-VCAM-1 D1-D4.The constructed plasmid was transformed into E.coli strain BL21(DE3).The VCAM-1 D1-D4 expressed in E.coli strain BL21 was detected by SDS-PAGE.On the other hand,the VCAM-1 D1-D4 cDNAs were inserted into the eukaryotic expression plasmid pIRES2-AcGFP1-Nuc.Recombinant plasmid pIRES2-AcGFP1-Nuc VCAM-1 D1-D4 was constructed successfully and transformed into Raji cells with Lipofectamine.The expression of VCAM-1 D1-D4 was highly upregulated in Raji cells.The construction and expression of VCAM-1 D1-D4 has been achieved successfully,which lays a foundation for further research on biological function of VCAM-1,and also provide experimental basis for further study of the B cell differentiation and development.