Construction, Expression And Identification Of Sflk1-IFN-γ Fusion Protein Encoding Recombinant Plasmid

Angiogenesis has a critical role in the growth, invasion and metastasis of tumors. It has been shown that tumor growth is generally limited to 1 to 2 mm~3 in the absence of a vascularized blood supply and inhibition of tumor angiogenesis is associated with suppression of tumor progression. Thus, antiangiogenic immunotherapy represents a novel modality for cancer treatment. Vascular endothelial growth factor (VEGF) receptor2 (VEGFR2, also known as flk1 in mouse) mediated VEGF signaling is the key rate-limiting step in angiogenesis. Recently, it is well established that soluble flkl (sflk1) can competitively inhibit VEGF binding to flk1, leading to an inhibition of tumor-induced angiogenesis and metastasis. IFN-γ is one of the major effector molecules in cell-mediated immunity, which plays important roles in the induction of CTL and differentiation of Th1 cells. Moreover, IFN-γ can directly inhibit the growth of a variety of tumor cells, and it also inhibits angiogenesis. Therefore, we constructed pcDNA3.1(+)/sflk1-IFN-γ recombinant plasmid, then selected for stably transfected CHO cell line expressing sflk1-IFN-γ fusion protein and identified the biological activities of such fusion protein.ObjectiveConstruction of the eukaryotic plasmid that harbors sflkl and mlFN-y fusion gene and expression of sflkl-IFN-y fusion protein in CHO cells. Selection for efficient and stable sflkl-IFN-y expression CHO cell line and identification of the biological activities of the fusion protein.To compare sflkl-IFN-y fusion protein with sflkl and IFN-y on their biological activities, the plasmids pcDNA3.1(+)/sflkl and pcDNA3.1(+)/IFN-y were also constructed and expressed in CHO cells.Method1. The eukaryotic expression plasmid pcDNA3.1(+)/sflkl-IFN-y was constructed by inserting sflkl and mIFN-y cDNA into pcDNA3.1(+) vector. The mIFN-y-encoding fragment was cloned into pcDNA3.1(+) vector to form pcDNA3.1(+)/IFN-y. pcDNA3.1(+)/sflkl was kept in our laboratory.2. COS-7 cells (a cell line from the kidney of an African green monkey) were transfected with recombinant plasmids pcDNA3.1(+)/sflkl-IFN-y, pcDNA3.1(+)/sflkl, pcDNA3.1(+)/IFN-y and pcDNA3.1(+) respectively. Culture supernatants were collected 48h later and used for identification of biological activity of IFN-y. Culture supernatants of pcDNA3.1(+)/sflkl-IFN-y, pcDNA3.1(+)/sflkl, pcDNA3.1(+)/IFN-y transfected CHO cells were used for identification of biological activity of sflkl. Protein expression of sflkl-IFN-y by transfected COS-7 cells was detected by ELISA and Western blotting.3. In order to get the CHO cell line that efficiently and stably express the interest protein, dhfr marker gene amplification strategy and limiting dilution were employed. Briefly, we co-tranfected pSV2-dhfr+ vector and recombinant plasmids pcDNA3.1(+)/sflkl-IFN-y, pcDNA3.1(+)/sflkl and pcDNA3.1(+)/IFN-y separately into CHO-dhfK cells and selected for stable and efficient sflkl-IFN-y fusion proteinexpression CHO cell line by adding increasing concentrations of MTX, finally obtained the cloned cell line by limiting dilution.Result1. Construction of recombinant plasmidsConstruction of pcDNA3. l(+)/sfikl-IFN-y or pcDNA3.1(+)/IFN-y was verified by double enzymatic digestion and DNA sequencing. The results were consistent with those expected, showing successfully construction of recombinant plasmids pcDNA3.1(+)/sflkl-IFN-y and pcDNA3.1(+)/IFN-y.2. Expression of sflkl and IFN-yELISA analyses showed that concentrations of sflkl and IFN-y in the culture supernatants of COS-7 cells transfected with pcDNA3.1(+)/sflkl-IFN-y were 20.85 ±2.48ng/ml and 1.08±0.09ng/ml respectively, sflkl in culture supernatants of COS-7 transfected with pcDNA3.1(+)/sflkl was 17.72 ± 1.83ng/ml, and the expression of IFN-y by pcDNA3.1(+)/IFN-y transfected COS-7 was 1.60 ± 0.14ng/ml, significantly higher than those expressed by pcDNA3.1(+) transfected COS-7 cells (sflkl=0 and IFN-y=0.014±0.007ng/ml). Western blotting analysis showed that sflkl appeared at about 115kDa and sflkl-IFN-y fusion protein appeared at 130 kDa in the lysate of COS-7 cells transfected with the corresponding recombinant plasmid, showing the correct size of sflkl and sflkl-IFN-y fusion protein.3. Identification of biological activity of sflkl-IFN-y fusion protein Supernatants of pcDNA3.1(+)/sflkl-IFN-y- and pcDNA3.1(+)/sflkl-transfectedCHO cells could inhibit rmVEGF164 binding to flkl which was expressed by H5V cells, leading to an growth inhibition of H5V cells. Supernatants of pcDNA3.1(+)/sflkl-IFN-y- and pcDNA3.1(+)/IFN-y-transfected COS-7 cells significantly enhanced the NK activity compared with those of pcDNA3.1(+)/sflkl-, pcDNA3.1(+)-transfectedCOS-7 and RPMI 1640. All of these suggest that the sflkl-IFN-y fusion protein owns the biological activities of both sflkl and IFN-y.4. Selection for efficient and stable sflkl-IFN-y expression CHO cell line We use a step wise increase method of MTX concentration to select for efficient and stable CHO cell line expressing sflkl-IFN-y fusion protein. sflkl-IFN-y expression level increased obviously as MTX concentration in the culture media increased gradually. After three rounds of limiting dilution, the clone of CHO-sflkl-IFN-y was obtained that secreted sflkl up to 229.32 + 10.70 ng/106cells/48h, and IFN-y up to 6.70±0.28ng/106cells/48h. In this way, the stable CHO cell line expressing high level of sflkl-IFN-y fusion protein was finally established.Conclusion1. Successfully constructed the eukaryotic expression plasmids pcDNA3.1(+)/sflkl-IFN-y and pcDNA3.1(+)/IFN-Y.2. sflkl-IFN-y fusion protein could be efficiently expressed by eukaryotic cells. Furthermore, it possesses the biological activities of both sflkl and IFN-y.3. Established stable CHO cell line expressing high level of sflkl-IFN-y fusion protein.