Preparation Of Recombinant Long-acting Human Interleukin-7-CTP Protein In CHO Cell And Research On Biological Activities

The human interleukin-7 (IL-7) gene was identified in 1988 and is located on chromosome 8ql2-13. IL-7 protein has 153 amino acids, containing three disulfide bonds, three N glycosylation and one O glycosylation.The amino acids sequence of human IL-7(a protein of 25-28 kDa produced by bone marrow stromal cells) and murine IL-7 (129 amino acids, a protein of 25 kDa) show 60%homology. The human IL-7 protein is 17 amino acids longer than the murine protein since the human IL-7 gene contains an additional exon,the fifth exon.IL-7 is mainly produced by non-hematopoietic stromal cells and epithelial cells,including fibroblastic reticular cells and T-cell areas of lymphoid organs, bone marrow stromal cells, liver and intestinal epithelial cells, endothelial cells, fibroblasts, keratinocytes,while a small part of IL-7 is produced by dendritic cells and macrophages cells. IL-7 exists in most organizations,even in brains,and the receptors of IL-7 are aboudant.The receptors of IL-7 are expressed on almost all of the peripheral T cells.Studies have shown that IL-7 can direct control and activate the signals of transduction through a IL-7R mediated feedback loop.Human IL-7 is an important factor of immunological regulation and has multiple immune-enhancing properties. It is important for proliferation during certain stages of B-cell maturation, T and NK cells survival, development and homeostasis. It can make hematopoietic stem cells into lymphoid progenitor cells.IL-7 is very important for T cell differentiation and keep memory T cells survival and plays a key role in the T cell homeostasis. IL-7 is important for the negative-positive selection of T cells during the T cell development. IL-7 make naive T cells activate and undergo many rounds of expansion as they differentiate into effector cells and improve the abundance of T-cell receptor (TCR), when the immune system is first challenged by exposure to antigen. Studies have showned that IL-7 enhanced the effector CD4+T cell and induced the effector T cell with the receptor of IL-7 to develope into long-term survival memory T cells. IL-7 can prevent apoptosis in the thymus ontogeny to maintain the memory T cell suvivial by up regulation Bcl-2 and Mcl-1 which provide anti-apoptotic signal and inhibition the appoptosis signals,such as Bax and BIM. IL-7 can support memory T-cell turn over,in lymphopenic mice,IL-7 supports the division of both CD4+ and CD8+ T cells.IL-7 is an important cytokine for B cell development,especially make pre-pro-B cell develope into B cell. Developing B-lineage cells proliferate in response to IL-7 by interacting with bone marrow stromal cells, which are the source of this cytokine. Following an in-frame V to (D)J recombination event, the successful expression of an Igp. chain leads to its assembly with the surrogate light chain and the signalling subunits Iga and IgP to form a pre-B cell receptor (pre-BCR). The pre-BCR promotes the generation and expansion of a population of large pre-B cells (also known as pre-BII cells), which remain dependent on IL-7 signalling.IL-7-induced dimerization of the CD127/IL-7Ra and yc subunits leads to a complex pattern of biochemical signaling involved JAK3/STAT5, PI3K/AKT, some Bcl-2 family members, and to a lesser extent RAS/MAPK and src family kinases.Some diseases or treatments induced immune system damage, such as the absence of CD4+T cells caused by HIV infection, Deletion and reduction of some blood cells after chemotherapy or radiation treatment. Several scientific researches launch to recovery the immune system, such as erythropoietin (EPO) which can remode red bood cells, granulocyte colony stimulating factor for remodeling center granulocyte and macrophages. IL-7 in animal experiments and clinical studies showed that it can enable immune system damage by infection or therapy to reconstruct and recovery. Immunogenicity of autoimmune antigens and tumor antigens is weak.IL-7 treatment can improve the immune response, in particular, can greatly increase the low affinity or weak immune response self. The biology of IL-7 make it a potential adjuvant for cancer immunotherapy.The CTP of HCG subunit that contains four 0 glycosylation fuse with other protein can not affect assembly,secretion, receptor binding affinity, or in vitro bioactivity.However, the in vivo potency and circulatory half life of the proteins containing CTP were substantially increased.Infections by HIV, HBV, HCV or TB are serious public health problems in China,even the world. The incidence of cancers has increased in the past decade,the rising trends will exacerbate the growing cancer burden associated with population expansion and aging. IL-7 as an alternative drug or adjuvant for the treatment of chronic viral infections or cancers,the application prospects of IL-7 is promising.So far,at least 27 clinical trials of IL-7 are carrying on, targeting to patient infected by HIV, HBV, HCV, TB and cancers.Therefore, we expressed the long-acting IL-7 protein and determined its activity. We fuse IL-7 with CTP and add a N glycosylation site at the C end of CTP.The codon of the sequence were optimizated and cloned into CHO cells.Positive cells were selected by antibiotics and MTX,the high expression cell were selected by Flow cytometry.IL-7-CTP were pruficated from the cell cuture supernant.The effect of IL-7-CTP protein on the proliferation of T and PBMC was studied.The gene expression of PBMC stimulated by IL-7-CTP and IL-7-RD was studied by the chip technology.The activity of IL-7-CTP in CTLL cells and the half-life in mice was determined.1.Expression of IL-7-CTP in CHO cellWe got the mRNA gene sequence of human IL-7 in GenBank and got the CDS sequence. We fused the CDS with the nucleic acid sequence of CTP protein by linker sequnce..CTP is carboxyl-terminal peptide of human chorionic gonadotropin β Subunit to C-terminal coding sequence,which contains four O glycosylations and one N glycosylation. The codon of IL-7-CTP gene sequences was optimizated by softwares and tools online. Eukaryotic expression vectors were constructed,such as pcDNATM5/FRT,pIRES-CD20-IL-CTP, PCI-IRES-CD20-IL-7-CTP. The expression plasmid was transfected into FLP-INTM-CHO or CH0-K1 and CHO/dhfr-cells, respectively,by Lipofectamine.The positive clones were selected by hygromycin B or G418 or nutritional deficiencies for 3-4 weeks. Hundreds of strains high production cells were sorted from tens of millions of positive cells by flow cytometry. Two strains of CHO-K1 tranfected by pIRES-CD20-IL-CTP can produce IL-7-CTP about 10mg/l/day.2. Purification of IL-7-CTP proteinCrude cell culture medium was collected and centrifμged to pellet whole cells and cells debris. Ammonium sulfate precipitation was used to selective the protein at 40-90% concentration,and then the protein precipitation is used to eliminate contaminants contained in FBS at the PH from 4.8 to 6.0.Then hydrophobic interaction chromatography,ion exchange,Blue Sepharose,Sephacryl S-200 molecular sieve chromatography were used to purification the IL-7-CTP,then the recovery of the different molecular weiht IL-7-CTP were mixed.The mixture show seven bands analysised by western blot. It was possible that 7 proteins were produced by glycosylation.The purity of this protein is about 10%.So the CNBr-CTP were used to purification the mixture and the recovery were filterd by 0.22 um filter and the quantity of IL-7-CTP were evaluated by anti-human IL-7 ELISA kits.We got 8.77 μg IL-7-CTP protein and it’s purity is 83.2%。3. In vitro activity of IL-7-CTPThe cytokine activity of the IL-7-CTP was determined in vitro in a cellular proliferation bioassay with the musine T lymphocyte cell, i.e. CTLL. An ED50 value of cell proliferation for IL-7-CTP proteins was obtained from plotting a dose response curve according to standard techniques, and determining the protein concentration that resulted in half-maximal response. The ED50 values of the fusion proteins is 0.90 ng/ml and active unit is 1.11×106U/mg,while the ED50 values of IL-7-RD is 0.635 ng/ml and active units is 1.58×106U/mg.PBMC proliferation was detected by flow cytometry and celltiter 96(?) non-radioactive cell proliferation assay when two kinds of IL-7 work on at 60 hours and 5 days after PHA-P activated. The results show that IL-7-CTP are stronger than IL-7-RD to induce the PBMC proliferate in same time and same concentration.4. Pharmacokinetics of IL-7-CTP proteins IL-7-CTP and IL-7-RD (IL-7 purchased from R&D Systems) were intraperitoneal injection to two groups of mice(average weight is 15g) with 2 μg/mice. Blood samples were obtained by retro-orbital bleeding at 0.5h,2 hs,6 hs,26.2 hs,30hs post-injection.Samples were collected in heparin-tubes to prevent clotting, and cells were removed by centrifμgation. The concentration of administered IL-7 was determined in blood serum at each time point by an ELISA specific for human IL-7. PK values were calculated with kinetical.The half-time and MRT of the IL-7-CTP is 25.67 hours and 36.27 hours,respectively,while the IL-7-RD is 5.79 hours and 8.17 hours.In summary, this study obtained hundreds of strains of stable CHO cells secreting IL-7-CTP and two cell strains’expression reached 10mg/l/day. A preliminary method of purifying IL-7-CTP was established. Some biological activities of IL-7-CTP protein were tested, such as half-life is 25.67 hours,ED50 is 0.9 ng/ml.The proliferation to activited PBMC and T cells of IL-7-CTP was tested and verified.