| Ieukocyte-associated immunoglobulin-like receptor- 1(LAIR-1, 9.1C3 and p40) are different names for the identical molecule. The cDNA encoding LAIR-1 was identified in 1997. which is located on human chromosome 19ql3.4. LAIR-1 belongs to the immunoglobulin superfamily(IgSF) with an Ig C2-1 domain in extracellular region and two ITIMs in cytoplasmic region. The ITIM can recruit the SHP-1/2 when bound with its ligand or cross-linked by corresponding antibody. Previous study on cellular distribution indicate that LAIR-1 is expressed mainly on the lymphocytes including T cells, B cells and NK cells. LAIR-1 plays the inhibitory role in the immunological response via its inhibition on activation signals. LAIR-2, the ancestor gene of LAIR-1 and its gene conjuncting to LAIR-1 gene in the leukocyte receptor complex(LRC), is a potential secretary protein, sharing 84% similarity in its Ig-like domain with LAIR-1. Recently, the mouse LAIR-1 gene was cloned in our lab.Epithelial cellular adhesion molecule(Ep-CAM) belongs to the adhesion molecule with two epithelial growth factor (EGF)-like domains in extracellular region and cystine-rich at its N-terminal. It has been demonstrated that Ep-CAM structure is unique compared with other adhesion molecules. Ep-CAM is broadly expressed on normal epithelials mediating the homophilic adhension as well as some neoplasm with epithelial origin, expression degree of which is closely related to tumor differentiation.The interaction of Ep-CAM with LAIR-1/2 was identified in 2001 and it was confirmed that the first EGF-like domain of Ep-CAM was involved in the recognition with LAIR family.The contents of this article can be divided into the following five parts.Firstly, Four new LAIR-1 isoforms were cloned from leukemia cell line HL-60 and Jurkat, named as L-1, L-2, H-l and H-2 respectively according to their origin and sizes. Among them, L-1, H-l and H-2 contain the complete open reading frame. Only L-1 and H-2 keep the Ig-like domain in the extracellular region. Because no similar genes were cloned from several PBMC of healthy donors, these isoforms may be related to the unusual of LAIR-1 mRNA alternative splicing in the leukemia cells.Secondly, the vectors for the expression of the eukaryotic(LAIR-1 -myc) and proeukaryotic(LAIR-1-GST) were constructed and the fusion proteins were expressed. The LAIR-1-myc in supernatant was purified with anti-c-myc monoclonal antibody affinity chromography and the LAIR-1-GST in bacteria lysate was purified with glutathione Sepharose. The polyclonal antibodies against LAIR-1 were prepared and identified.Thirdly, the cDNA of LAIR-2 was cloned from the PBMC of the patient with HFRS. The vector construction and fusion protein expression LAIR-2-myc and LAIR-2-GST , as well as the preparation of monoclonal antibodies against LAIR-2, were carried out using the same procedure as above. After the routine technique of immunization and cell fusion, 4 strain monoclonal antibodies against the protein was got. The titers of mAb in ascites reached to 10-6. The sandwich ELISA for detecting LAIR-2 was prepared with the limitation of about 5ng/mL.Fourthly, cDNA coding Ep-CAM was obtained by RT-PCR from neoplasm of breast and rectal carcinoma. The Ep-CAM-GST fusion protein was expressed as before. 15 strains mAb was obtained. The characteristic of the mAbs was identified by flowcytometry and immnohistochemistry technique. Interestingly, the different mAb staining pattern in immunohistochemistry was found. Among them, 2A9 mAb only stained the membrane of the gland epithelials, while 4B11 mAb can stain the membrane nucleic membrane of epithelials and the inflammation cells around them as well.Finnaly, epitopes involving the interaction of LAIR family with Ep-CAM was investigated with ELISA and confocal technique. It was found that the Ep-CAM epitopes recognized by LAIR-1 and LAIR-2 was distinct by ELISA blocking experiment. 2A9 mAb could block the binding of both LAIR-1 and LAIR-2 with Ep-CAM. 2D5 mAb, 2D7 mAb and 2H6 mAb could inhibit t...
|
PDF Full Text Request