Human Interleukin -23 Receptor (hil-23r) Gene Expression And A Preliminary Study

The human interleukin 23 receptor(hIL-23R),a new cytokine receptor reported in 2002,is a member of the hemopoietic cytokine receptor family,related to IL-12Rβ2 and gp130,IL-23R and IL-12Rβ1 together comprise the IL-23 receptor complex on IL-23-responsive cells,The IL-23R protein is comprised of an extracellular domain,a single transmembrane domain and a 252 amino acid cytoplasmic domain,The extracellular domains of IL-23R contains a signal sequence,an N-terminal Ig-like domain and two cytokine receptor domains,related to corresponding domains of IL-12Rβ2,In contrast to IL-12Rβ2,IL-23R does not contain three transmembrane-proximal fibronectin typeⅢdomains.There are seven potential N-linked glycosylation sites in hIL-23R.The transmembrane-proximal cytokine receptor domain of hIL-23R contains a sequence (WQPWS) similar to the cytokine receptor signature WSXWS motif.The IL-12Rβ1 of IL-23R complex could bind to IL-23p40 subunit of IL-23 and IL-23R engaged signal transduction.IL-23 and its receptor are structurally and functionally related to IL-12 and its receptor.Thus,many studies about IL-23 and its receptor is based on the functional studies on IL-12 and its receptor.It has been showed that IL-23 has anti-tumor activity.In addition, IL-12Rbeta2 gene also functions as a tumor suppressor in human B cell malignancies.But the role of hIL-23R in anti-tumor process is currently unknown.Our previous study found that the differential expressions of spliced variants of IL-23R are presented in many tumor cells,implying that aberrant expression of hIL-23R may be associated with cancer development.The aims of this study are to clone the full length of hIL-23R gene,and to investigate its expression in E.coli and human normal cells.Firstly,we cloned the CDS of hIL-23R gene from human peripheral blood mononuclear cells by RT-PCR,constructed the eukaryotic and prokaryotic hIL-23R protein expression vector pCMV-Myc-hIL-23R and pGEX-4T-1-hIL-23R.Secondly,we studied the eukaryotic expression of hIL-23R,tested its expression in HEK 293T cells by RT-PCR and Western blot analysis,defined the subcellular location of hIL-23R protein in HEK 293T cells and COS-7 cells;tested its contribution to cell cycle and cell apoptosis by Flow Cytometry.Thirdly,we study the prokaryotic expression of hIL-23R protein,successfully induced GST fusion protein's expression in E.coli BL21(DE3),which can be used for specific antibodies' production and Western blot was performed to confirm the expression of hIL-23R fusion protein.Finally, we did PCR using the sequencing correct vector pMD-19 T-hIL-23R as template,and got the different regions of hIL-23R,constructed the prokaryotic extracellular domain of human IL-23R protein expression vector,and the prokaryotic intercellular domain of human IL-23R protein expression vector,respectively;the purification of hIL-23R protein from bacteria may be useful for the production of specific antibody against human IL-23R protein in the future.In conclusion,we successfully constructed the eukaryotic and prokaryotic hlL-23R protein expression vector pCMV-Myc-hlL-23R and pGEX4T-1-hlL-23R,study its expression in E.coli and HEK 293T cells,tested the contribution of hlL-23R to cell cycle and cell apoptosis by Flow Cytometry.successfully constructed the extracellular and intercellular region of hlL-23R expression vector,which may contribute to the therapeutic purpose and functional studies of hlL-23R in the future.Our study built up the basis for the further functional characterization of hlL-23R.and its relativity with tumor.