| Objective: CD40 single-chain antibody(CD40 single-chain variable fragment,CD40 Sc Fv)was prepared in vitro to evaluate the effect on accelerating the secretion of IL-12 by dendritic cells(DCs).The proliferation of T helper lymphocytes 1(Th1)was induced by IL-12,which increased the secretion of cytokines with anti-tumor effect and thus plays an anti-tumor effect.For its further application in clinical provide experimental basis.Methods: 1.Preparation of CD40 Sc Fv protein: the target gene CD40 sc Fv was synthesized by PCR amplification according to the previously obtained p CANTAB5E-CD40 Sc Fv phagemid.Then cutting technology was used to construct p ET28a-CD40 Sc Fv plasmid.The recombinant plasmid was transformed into Escherichia coli Rosetta.The CD40 Sc Fv protein was purified by Protein affinity chromatography.Then the purified protein was measured by BCA protein content,and was identified by SDS-PAGE and Western blot.2.Study of CD40 Sc Fv inducing Th1 cell polarization in vivo: First,Establishment of Balb/C mice model of right axillary tumor in mice with breast cancer cell line 4T1.According to different medicine treatment,the tumor bearing mice were divided into 3 groups: CD40 Sc Fv group,CD40 m Ab group and NS group.Observing the changes of tumor volume of mice.The mice were killed and the tumor tissue was separated.Then the concentration of IL-12 in supernatant of tumor tissue was measured by ELISA.Th1 and Th2 in TILs were measured by flow cytometry.3.Study of CD40 Sc Fv inducing Th1 cell polarization in vitro: Cultured DC in vitro and DC tumor antigens was measured.According to different conditions of activation,DC was divided into 3 groups: CD40 Sc Fv group,TNF-positive control group,negative control group.DC surface molecules were measured by flow cytometry.CD4+T lymphocytes were separated from mouse spleen in mice,then the DC were co cultured with CD4+T lymphocytes.IL-12 concentration in the supernatant was measured by ELISA.Th1 and Th2 in were measured by flow cytometry.Results:1.pET28a-CD40 ScFv recombinant plasmid was Constructed succseefully.CD40 Sc Fv protein was right when identificated by SDS-PAGE and Western blot.The molecular size of CD40 Sc Fv protein is about 27 KDa,the volume was about 1.12mg/ml.2.The concentration of IL-12 in the supernatant of tumor tissue in group CD40 Sc Fv was(396.27 ± 48.13)pg/m L,significantly higher than that of group NS(P<0.05),but there was no significant difference between group CD40 m Ab agonist and CD40 Sc Fv group(P > 0.05);The ratio of Th1/Th2 cells in CD40 Sc Fv group the mice in the tumor microenvironment is(6.32 ± 0.87),significantly higher than that of group NS(P < 0.05),but there was no significant difference between group CD40 m Ab agonist and group CD40 Sc Fv(P > 0.05).3.Experimental group CD40 Sc Fv culture supernatant for IL-12 concentration(674.37 ± 73.59)pg/m L,significantly higher than the group negative control(P < 0.05),but there was no significant difference between TNF-alpha positive control group and CD40 Sc Fv group(P > 0.05);The ratio of Th1/Th2 cells the CD40 Sc Fv group is(9.98 ± 1.78),significantly higher than the negative control group(P < 0.05),and but there was no significant difference between TNF-alpha positive control group and CD40 Sc Fv group(P > 0.05).Conclusion : 1.p ET28a-CD40 Sc Fv recombinant plasmid was successfully constructed.2.After the recombinant plasmid was transformed into Rosetta,the expression of CD40 Sc Fv protein could be stably expressed by IPTG.3.CD40 Sc Fv can reverse the imbalance of Th1/Th2 cells in tumor microenvironment,and enhance the anti-tumor effect.4.CD40 Sc Fv can induce the secretion of IL-12 by DC in vitro,and then induce the polarization of Th1 cells. |
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