| Objective: To construct a prokaryotic expression vector of agonistic CD40 sc Fv and obtain agonistic CD40 sc Fv in high prurity.Then,to verify natural killer(NK)cell stimilulating effect and the antitumor effect of agonistic CD40 sc Fv in vitro and vivo.Methods: A prokaryotic expression vector of agonistic CD40 sc Fv was constructed.Expression of agonistic CD40 sc Fv protein was inducted using IPTG and protein was purified by affinity chromatography.In vitro,murine dendric cells(DC)were induced and cultured in vitro and then were stimulated utilising agonistic CD40 sc Fv.Expression of CD80,CD86 and MHC-II on DCs were detected by flow cytometry.Concentration of IL-12 in DCs culturing supernatant was detected by ELISA.Murine NK cells were isolated by immunomagnetic beads and then were stimulated by DCs with its culturing supernatant which were stimulated by agonistic CD40 sc Fv before.Concentration of IFN-? in culturing supernatant of NK cells was detected by ELISA.T6-17 cells were cocultured with stimulated NK cells.T6-17 cell apoptosis was dected by WST-8 to reflect NK cell cytotoxity.In vivo,T6-17 cell tumor model was constructed in Balb/c nude mice.Agonistic CD40 sc Fv was injected intratumorallly and data of tumor growth was recorded.Expression level of IL-12 and IFN-? in tumor homogenate was detected by ELISA.Tumor infiltration lymphocytes(TIL)were extracted,DX5 positive cells in TIL were detected by flow cytometry.NKG2 D expression levels of NK cells were detected by immunological histological chemistry staining.Results: A prokaryotic expression vector of agonistic CD40 sc Fv was succesfully constructed,which was verified by gene sequencing.Expression of target protein was successfully inducted and purified,which was about 27 k Da on SDS-PAGE electrophoresis.Purified protein was verified by Western Blot of histag.The concentration of purified protein is 1.122mg/ml.Murine DCs was succesfully induced in vitro.Expressions of MHC-II,CD80 and CD86 on CD40 sc Fv treated DCs were significantly elevated,respectively were 90.49%±3.77%(P=0.000),87.99%±3.35%(P=0.000)and 94.07%±3.13%(P=0.010).Concentration of IL-12 in DCs culturing supernatant of CD40 sc Fv group was 555.86±40.48 pg/ml,which was significant higher than control group(P=0.000).Concentration of IFN-? in NK cells culturing supernatant of CD40 sc Fv group was 296.71±24.62 pg/ml,which was significant higher than control group(P=0.015).Killing rate of NK cells of CD40 sc Fv group was also sigficant higher than control group with a killing rate of 72.23%±3.99%(P=0.004).T6-17 cell tumor model was succesufully constructed in Balb/c nude mice.Agonistic CD40 sc Fv inhibited tumor growth in vivo with a significant decrease of final tumor volume(P=0.000).Concentration of IL-12 and IFN-? in tumor mircroenvironment treated by agonistic CD40 sc Fv was 188.801±32.718 pg/ml and 121.428±30.994 pg/ml respectively,which were significantly higher than control group(P=0.023 and P=0.006).Ratio of CD3-DX5+ cells ratio in TIL of mice treated by agonistic CD40 sc Fv was 19.15%±2.24% and was significantly higher than control group(P=0.002).NKG2 D expression in tumor tissue of CD40 sc Fv group was also siginificanly higher than control group with a PEI% of 8.18%±2.01%(P=0.005).Conclusion:(1)A prokaryotic expression vector of agonistic CD40 sc Fv was constructed.Agonistic CD40 sc Fv protein was inducted and then purified successfully to obtain agonistic CD40 sc Fv in high purity.(2)Agonistic CD40 sc Fv upregulated expression of MHC-II,CD80 and CD86 on DCs in vitro.It also increased IL-12 secretion of DCs.Agonistic CD40 sc Fv stimulated NK cells through stimulating DCs,increase IFN-? secretion of NK cells and enhance its cytotoxity.(3)Agonistic CD40 sc Fv inhibited T6-17 tumor growth in vitro,increased level of IL-12 and IFN-? in tumor microenvironment,convened NK cell into tumor microenvironment and up-regulated expression of NKG2 D. |
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