Stable Expression And Development Of Osteoclast Inhibitory Lectinrelated Protein2(OCILRP2) In CHO Cells

BackgroundThe C-type lectin-like receptors such as NKG2Dand CD94/NKG2emerged as a new category of Tcell costimulatory molecules due to their demonstrated ability to costimulate T cell proliferation andcytokine secretion. The C-type lectin family is characterized by carbohydrate recognition domain (CRD).Their CRD domains are named C-type lectin-like domain (CTLD).Experiments has showing that C-type lectin-like molecules, including NK cell receptorsCD94/NKG2A, NKG2D, NKRP1family member and CD69, play important roles in innate as well asadaptive immune responses. The C-type lectin receptor members serve diverse functions in the immunesystem by modulating pathogen recognition, NK and T cell stimulation, cell trafficking, and boneformation.Osteoclast inhibitory lectin-related protein2(osteoclast inhibitory lectinrelated protein2, OCILRP2)is a member of osteoclast inhibitory lectin (OCIL) family， which belongs to type Ⅱ transmembraneglycoprotein, with a smaller intracellular area and the extracellular domain containing a C-type lectindomain. OCILRP2is a novel C-type lectin. Mouse OCILRP2gene is located in the NK gene complex ofchromosome6. OCIL family includes OCIL (Clrb), OCILRP1(Clrd) and OCILRP2(Clrg)3genes, theextracellular region is highly homologous (about80%).OCILRP2mainly expressed in the immune system, which is high expression in the spleen and thethymus, but can not be tested in the brain, heart, liver, kidney, testis, lung, placenta and muscle by PCR.Itwas found that OCILRP2is highly expressed in B cells and dendritic cells (DC) and is lowly in unactivated T cells, but is up-regulated in activated T-cells. OCILRP2as a member of the NK cell receptorprotein1(NKRP1) family is a ligand ofNKRP1f. Studies have shown that OCILRP2also is a member ofT-cell co-stimulatory molecules and plays an important role in the activation of T cells.The novel couple of C-type lectin receptor and ligand is involved in T cell costimulation. The receptor,OCILRP2, is rapidly induced following T cell activation and maintained at a substantial level of up to72h.The ligand, NKRP1f, is predominantly expressed on dendritic cells (DC). The soluble OCILRP2-Igblocking protein significantly suppresses specific antigen-stimulated T cell proliferation as well as IL-2 secretion both in vitro and in vivo; conversely, NKRP1f-expressing antigen presenting cells (APC) enhanceB7.1/CD28-mediated costimulation for T cell proliferation through interaction with OCILRP2. But theaction mechanism of OCILRP2in the T-cell co-stimulatory is not clear.ObjectiveTo purify and to identify OCILRP2-Ig Fc fusion protein stablely expressed in Chinese hamster ovary(CHO) cells; To develop the polyclonal antibody of the OCILRP2to immune rabbit by OCILRP2-Ig Fcfusion protein.MethodsExpression vector OCILRP2-Ig Fc were transfected into the mammalian cell of CHO cells withLipofectamine2000. The selective medium with G418was then employed to select the positive colony.After repeating cloning culture and ELISA detection, the positive cell clones with stable expressionOCILRP2-IgFc fusion protein were obtained. The cells was intermediate cultured in cell culture flask withthe serum free medium(SFM) for five to seven days, culture supernatant (SCS) was harvested. After thesample was hypothermia and high speed centrifuged, the sample was filtrated with0.45micron filtermembrane. After purified by proteinA affinity chromatography, OCILRP2-IgFc was studied by BCA,SDS-PAGE,Western blot, ELISA, and the mass chromatographic analysis to determine or assess itsconcentration，molecular weight, purity, antigen specificity and so on.With the ELISA and flow cytometry,we analyzed the biologic activity of the fusion proteinOCILRP2.The male New Zealand rabbits were immuned to prepare the anti-OCILRP2antiserum by theOCILRP2-IgFc fusion protein with the Freund’s complete adjuvant(CFA) or the incomplete Freund’sadjuvant(IFA) emulsification;Anti-OCILRP2polyclonal antibodies (PcAb) were purified by the method ofthe saturated ammonium sulfate, Protein G affinity chromatography.ResultsCHO cell clones stably expressing OCILRP2-IgFc fusion proteins were obtained and the fusion protein was available from the supernatant of cell culture media after purified by protein A affinitychromatography. Two protein main bands with molecular weight about55kDa and40kDa were found in theSDS-PAGE. Two protein specifically bounds with goat anti-human IgFc polyclonal antibody labeled HRP.The mass spectrometry authenticated two main bands all including the amino acid sequences ofOCILRP2and hIg Fc tag.The soluble OCILRP2-IgFc fusion protein significantly suppresses T cell proliferation as well as IL-2secretion and OCILRP2expressionThe specific reactions were confirmed between the fusion protein and the polyclonal antibody by theELISA and Western blot.ConclusionRecombined OCILRP2-IgFc fusion protein with block function and antigen specificity is successfullyand stably expressed in CHO cells. The polyclonal antibody of OCILRP2-Ig Fc fusion protein weredeveloped.