The cells were resuspended in DMEM containing 1% FBS at a density of 5 × 105 cells per milliliter. The cell suspensions (100 μl) were seeded into the upper chambers, and 600 μl of DMEM medium containing 10% FBS and 10 μM VLP H1 or VLP H2 was added to the lower chambers. The cells were allowed to invade for 12 h in a CO2 incubator, fixed, stained, and
quantitated as described previously . Results Expression and purification of fusion proteins RGD-IFN-α2a (300)-core, RGD- core-IFN-α2a(300), RGD-IFN-α2a-core, and RGD-core-IFN-α2a fragments were amplified using pMD-RGD-IFN-α2a (300)-core, pMD-RGD- core-IFN-α2a(300), pMD-RGD-IFN-α2a-core, and pMD-RGD-core-IFN-α2a as templates and subcloned into the pFastBacHTb-EGFP via BamH1/EcoRI sites and produced pFastBacHTb-RGD-IFN-α2a (300)-core (pH1), pFastBacHTb-RGD-core-IFN-α2a(300) (pH2), pFastBacHTb-RGD-IFN-α2a-core
(pH3), and pFastBacHT Saracatinib clinical trial b-RGD-core-IFN-α2a (pH4). The expression vectors pH1, pH2, pH3, and pH4 were confirmed on an agarose gel after double digestions with BamHI and EcoRI (Figure 1B,C) and further Selleckchem Nutlin-3a confirmed by DNA sequencing. Finally, the successfully constructed expression vectors pH1, pH2, pH3, and pH4 mediated the insertion of genes into the AcBacmid by Tn7-mediated transposition to generate AcH1, AcH2, AcH3, and AcH4 bacmids, respectively (Figure 1A). These recombinant bacmids were introduced by transfection into Sf9 cells to produce the recombinant proteins His-H1, His-H2, His-H3, and His-H4. The fusion proteins were purified from the supernatants of cell lysates using Ni-NTA affinity resin under click here native conditions. Intense bands corresponding to the molecular weights of the expected proteins are shown: 59.4 kDa for His-H1 and His-H2; 71.9 kDa for His-H3 and His-H4; the concentration of His-H1 and His-H2 is higher than the His-H3 and His-H4 (Figure 1D). Identification of virus-like particles To identify the impurities in the VLP
H1 and VLP H2 preparation after crude purification with successive sucrose gradients, various analyses were performed. First, confirmation of protein identity in the VLP preparation was performed by immunoblotting using HCV core-specific monoclonal antibodies (Figure 1G,H). In addition, EM analysis of the protein was performed and revealed spherical VLPs of 30 to 40 nm in size (Figure 1E,F). RGD- core-IFN-α2a fusion protein specifically binds with cancer cell line RGD (arginine-glycine-aspartic acid) can specifically bind with αvβ3 integrin, which is highly expressed on the cancer cell surface. The recombinant RGD- core-IFN-α2a protein was expressed and purified in Sf9. As expected, the recombinant RGD- core-IFN-α2a can specifically bind breast cancer cells MDA-MB231 and colon cancer cells HCT116 (data not shown) but do not bind normal cells such as normal human embryonic kidney cell 293 T.