Generation Of Recombinant Mouse MdsNKG2D-IL-21 Protein And Characterization Of Its Anti-tumor Activity Against Xenografted Colon Carcinoma Of Mice

DsNKG2D-IL-21 fusion protein composed by two of NKG2D extracellular domains and a IL-21 domain. It’s NKG2D can be targeted to identify the tumor cell surface MICA molecules, and it’s IL-21 promotes the function of NK cells and CD8+T cells, thus play an anti-tumor effect. Colon cancer is cancer of the colon mucosa epithelium, although can be eased by conventional postoperative chemotherapy or surgery, it still easy to relapse and metastasis. Given our previous research has confirmed that mouse dsNKG2D-IL-15 fusion protein can effectively enhance NK cell function in vitro and significantly inhibit the growth of colon cancer in vivo, IL-21 has a high degree of homology with IL-15 in structure and has a more remarkable anti-tumor function. In this study we aimed to generated the dsNKG2D-IL-21 fusion protein, then preliminarily observed effect of the protein on function of NK cells and CD8+T cells and analysis its anti-colon cancer activity in vitro and in vivo.Objective:To observe the mouse dsNKG2D-IL-21 fusion protein’s anti-tumor activity in vivo, we attempted to generate the homology mouse dsNKG2D-IL-21 fusion protein with the human source, so that to directly support the anti-tumor activity of hdsNKG2D-IL-21 fusion protein in vivo.Methods:(1) Production and Identification of mouse dsNKG2D-IL-21 fusion proteinsAt first, preparing a small amount of our previous recombinant plasmid pET28a-mdsNKG2D-IL-15, then removed the IL-15 fragment by using the restriction enzyme. The gene sequence of mouse Interleukin-21 was amplified by polymerase chain reaction (PCR) respectively. The gene fragment was inserted into pET28a-dsNKG2D vector, so a recombinant pET28a-mdsNKG2D-IL-21 prokaryotic vector was achieved. Next, the recombinant pET28a-mdsNKG2D-IL-21 vector was transformed into E.Coli (BL21), and the fusion protein was expressed as inclusion body induced by IPTG. Lastly, the recombinant dsNKG2D-IL-21 protein was purified and renatured successfμlly. NKG2D domain and IL-21 domain of the recombinant mdsNKG2D-IL-21 protein were respectively identified with NKG2D mAb and IL-21 mAb by using Enzyme-linked Immunosorbent Assay (ELISA) and Western Blot Assay. Whether mdsNKG2D-IL-21 protein can recognize RAE-1 molecule on YAC cells or not was detected by flow cytometry (FCM) (using Ba/F3 cells which do not express RAE molecule as a negative control)(2) Effect of mouse dsNKG2D-IL-21 fusion protein on NK cells and CD8+T cells function and its anti-colon cancer activity analysisAfter mouse spleen-derived lymphocytes were co-cultured with the recombinant mdsNKG2D-IL-21 protein overnight in vitro, expressions of CD69 and CD107a on NK cells or CD8+T cells were measured by FCM. IFN-y, perform and granzyme B production of NK cells was examined by an intracellular staining assay. In addition, Mice were injected mdsNKG2D-IL-21 protein for 24 hour to detect the change of percentage and activity of NK cells and CD8+T cells in vivo with the same method as above. At last, a mouse model of colon cancer was established in BALB/c mice by injecting CT-26 cells subcutaneously. On day 7, we separated the mice into two groups at random, PBS as control, mdsNKG2D-IL-21 protein. During 3 weeks, we observed and recorded the growth of tumor of tumor-bearing mice. On day 21, the change of percentage and activity of NK cells and CD8+T cells were analyzed by FCM.Results: (1) A recombinant expression vector pET28a-dsNKG2D-IL-21 was constructedsuccessfully, and it was strong expressed in E. Coli (BL21). Meanwhile, the recombinant mdsNKG2D-IL-21 protein was successfully purified and renatured to be used in further study. The renatured recombinant dsNKG2D-IL-21 protein could specifically bind to NKG2D mAb and IL-21 mAb. In addition, this protein could recognize RAE-1 molecule on RAE-1 positive tumor cells. (2) The recombinant mdsNKG2D-IL-21 protein could induce the expression of CD69 onNK cells and CD8+T cells whether in vivo or in vitro, enhance the killing of YAC cells with the expression of CD107a and facilitate production of the IFN-y, perform and granzyme B. The recombinant mdsNKG2D-IL-21 protein could promote the cytotoxic activity and proliferation of NK cells and CD8+T cells. In addition, The recombinant mdsNKG2D-IL-21 protein could inhibit colorectal cancer cells growth in vivo ultimately. The expression of CD69 on NK cells and CD8+T cells raised in the tumor-bearing mice treated with dsNKG2D-IL-21 protein, production of perform and granzyme B also a slight increase. Conclusions:The recombinant mdsNKG2D-IL-21 protein was successfully generated. It promoted NKcells and CD8+T cells activation and inhibited colorectal cancer cells growth in mice. In a word, our study supports that our mouse dsNKG2D-IL-21 protein provide important basis for the anti-tumor action in vivo of human dsNKG2D-IL-21 protein.