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目的对pSG.SS.Flk-1265-2493.C3d3.YL及pSG.SS.Flk-1265-2493.YL真核表达质粒的免疫效应进行初步研究。方法构建pSG.SS.Flk-1265-2493.C3d3.YL及pSG.SS.Flk-1265-2493.YL真核表达质粒后,48只BALB/c小鼠随机均分为疫苗组、疫苗对照组、质粒对照组、空白对照组(12只/组),分别肌肉注射接种此2种质粒、pSG.SS.YL质粒和PBS。免疫后动物:①每组6只取脾脏分离细胞,MTT法测定经Flk-1蛋白刺激后的增殖效应,乳酸脱氢酶释放法测定对培养小鼠肺微血管内皮细胞的杀伤作用;②其余6只动物于不同时间点断尾取血分离血清,ELISA法测定抗Flk-1抗体水平的动态变化。结果经Flk-1蛋白刺激,疫苗组小鼠脾淋巴细胞的增殖效应明显较强,刺激指数显著高于其他3组(P均<0.01),对靶细胞(MVECs)的杀伤活性在不同效/靶比时均显著高于其他3组(P均<0.01)。疫苗组动物血清抗Flk-1抗体比对照疫苗组抗体阳性反应出现早2周,二者峰值效价均出现在5周(血清稀释度:1∶3840vs1∶60),前者12周时仍保持较高水平(1∶960),后者10周时抗体阳性反应已消失。结论C3d3的连接有效增强了Flk-1265-2493的免疫原性。
Objective To study the immune effect of eukaryotic expression plasmids pSG.SS.Flk-1265-2493.C3d3.YL and pSG.SS.Flk-1265-2493.YL. Methods After constructing eukaryotic expression plasmids pSG.SS.Flk-1265-2493.C3d3.YL and pSG.SS.Flk-1265-2493.YL, 48 BALB / c mice were randomly divided into vaccine group, vaccine control group , Plasmid control group and blank control group (12 mice / group). The two plasmids, pSG.SS.YL plasmid and PBS were injected intramuscularly. Immunized animals: (1) 6 spleens of each group were used to separate the cells; MTT assay was used to measure the proliferation effect after stimulation with Flk-1 protein; and the killing effect on the cultured mouse lung microvascular endothelial cells was measured by lactate dehydrogenase release method; Only the animals at the different time points were taken from the tail of the blood to separate the serum, and the ELISA method was used to measure the dynamic changes of anti-Flk-1 antibody level. Results The proliferation of splenic lymphocytes in the vaccine group was significantly stronger than that in the other three groups (P <0.01). The killing activity of the target cells (MVECs) in different efficiency / The target ratio was significantly higher than those in the other three groups (all P <0.01). The antibody titer of anti-Flk-1 antibody in serum of vaccine group was two weeks earlier than that of control vaccine antibody, the peak titers of both groups appeared in five weeks (serum dilution: 1:3840vs1:60) High level (1: 960), the latter 10 weeks the antibody positive reaction has disappeared. Conclusions C3d3 ligation effectively enhances the immunogenicity of Flk-1265-2493.