Construction, Expression, Biological Functions Of MIP-2γ/GM-CSF Fusion Protein

Cytokine is a kind of proteins of high activity and multiple functions, produced by hematopoietic cells and immune cells. Cytokine can mediate the proliferation, differentiation of its target cells, and mediate some physical and pathological processes, such as hematopoiesis regulation and immunoresponsiveness. Preparation of cytokine fusion protein is one of the hot topics because cytokines fusion protein constructed by genetic engineering, elicits the biological effects of both its component cytokines and represents a better activity of delivering the two independent components. Up to now, many kinds of fusion proteins have been prepared. Some of those have been used in clinical trials such as GM-CSF/IL-3 fusion proteinIn our previous study, we cloned and characterized a novel CXC chemokine without ELR motif from the human DC cDNA library. The chemokine was named as MIP-2γ because of highest homology with macrophage inflammatory protein (MIP)-2α/β. MIP-2γ is an effective chemoattractant for some immune and hematopoietic cells ,such as immature DC and neutrophils. It can also regulate the hematopoiesis . Interestingly, MIP-2γ exhibits an enhancing effect on the proliferation and differentiation of the myeloid progenitor cells and the megakaryocytic progenitor cells together with many hematopoietic cell growth factors .As we know, GM-CSF was demonstrated to promote the ability to multiple hematopoiesis growth ability. GM-CSF can improve the proliferation and differentiation ability of the many linages of hematopoietic progenitor cells , enhance the effect on phogocytosis of neutrophils and monocytes. GM-CSF can also induce improve maturation of dendritic cells(DC)and enhance the antigen presenting ability of DC.GM-CSF has been used to promote hematopoietic recovery in cancer patients who received chemotherapy or radiotherapy. These data suggest that the biological functions of MIP-2γand GM-CSF are complementary each other. Weshould select MIP-2γand GM-CSF to construct a new fusion protein. The MIP-2γ/ GM-CSF fusion protein may attract or recruit more immune cells and hematopoietic cells, and then activate or enhance the functions of those cells. So the new fusion protein may have better activity than single cytokine and may regulate hematopoesis, and immune response more efficiently.In the present study, we constructed expression vector encoding MIP-2γ/GM-CSF fusion protein. After MIP-2γ/GM-CSF fusion gene vector was transfected into COS-7cells, we collected the supernatants of COS-7cells transiently expressing MIP-2γ/GM-CSF fusion protein. Then we investigated the hematopoietsis and chemotaxis activity of the fusion protein.First, we constructed hMIP-2γ gene expression vector by ligating hMIP-2γ with pcDNA3.1/myc-His(-)A eukaryotic gene vector. Then, MIP-2γ gene and hGM-CSF gene were linked via a flexible fragment connect which was coded by the 947-983bp sequences of pcDNA3.1/myc-His(-)A vector. By the same way we constructed MIP-2γ/mGM-CSF fusion expression vector. The two fusion gene vectors were confirmed by the NheⅠ,HindⅢ restriction enzyme analysis and auto sequencing analysis. MIP-2γ/ hGM-CSF fusion gene was transfected into COS-7 cells. For transient expression the fusion protein ,after 48 hours, we obtained the MIP-2γ/ hGM-CSF fusion protein from the cell supernatants . Anti hGM-CSF mono colony antibody was used in Western blot assay for detecting the expression of fusion protein . A remarkable 28 KD band of MIP-2γ/ hGM-CSF fusion protein was observed. Then, by human GM-CSF immunoassay we determined the GM-CSF concentration of the fusion protein in the supernatants by ELISA and GM-CSF was detected. 6.8ng/ml. According to the ratio of h MIP-2γand hGM-CSF, we predicted the concentration of the fusion protein as about 10.4ng/ml. Successively, we investigated the biological functions of the MIP-2γ/hGM-CSF fusion protein. As a hGM-CSF dependent human erythrolrukemic cell line, MO-7e cells were employed to determine GM-CSF activity of MIP-2γ/hGM-CSF fusion...