日本血吸虫22.6kDa抗原基因序列中免疫抑制片段的确定

发布时间：2018-06-08 16:04

本文选题：日本血吸虫 + Sj22.6kDa抗原　；参考：《南京医科大学》2008年硕士论文

【摘要】： 【目的】研究日本血吸虫22.6kDa(Sj22.6)抗原基因序列中是否存在免疫抑制性片段。【方法】依据已经报道的免疫抑制性序列的结构特征,从编码Sj22.6kDa抗原的基因序列中选取若干长度均为20～30bp的寡脱氧核苷酸(oligodeoxynucleotide,ODN),人工合成这些短的DNA序列,以目前国际上公认的对小鼠具有很强免疫刺激作用的ODN CpG1826作为刺激剂,用于后续实验,并设立对照。分离正常小鼠脾脏单个核细胞进行体外培养,将不同ODN分别与CpG 1826共同加入细胞培养体系,同位素~3H-TdR掺入法测淋巴细胞增殖实验观察各组细胞增殖水平的变化,以筛选出抑制性ODN并观察抑制性ODN作用的量效关系和时间效应关系。以刀豆蛋白A(Concanavalin A,ConA)做刺激物,~3H-TdR掺入法测淋巴细胞增殖实验观察抑制性ODN对ConA诱导的细胞增殖的影响。采用酶联免疫吸附试验(ELISA)观察抑制性ODN在体内和体外分别对CpG 1826引起的Th1型细胞因子IFN-γ和IL-12分泌的影响。采用流式细胞术检测抑制性ODN对细胞结合和摄取CpG 1826的影响。最后采用半定量PCR的方法检测抑制性ODN对CpG 1826的特异性受体Toll样受体9(TLR9)mRNA表达水平的影响。【结果】(1)淋巴细胞增殖试验结果显示,Sj22.6kDa抗原基因序列中的ODN F546和F311能抑制CpG 1826诱导的小鼠脾脏淋巴细胞体外增殖,在各ODN终浓度均为1μM时,ODN F546和F311抑制率分别为87%和11%。(2)淋巴细胞增殖试验结果显示,当CpG 1826的量一定时,ODN F546和F311的抑制作用随着它们量的增多而增强。当与CpG 1826的摩尔比为1∶1时,ODN F546或F311的抑制作用在同时或先于CpG 1826加入时最强。(3)酶联免疫吸附实验结果显示,在体外和体内实验中,ODN F546均显著降低CpG 1826诱导的小鼠脾细胞产生的Th1型细胞因子IFN-γ和IL-12的分泌量。(4)流式细胞术检测结果表明,ODN F546使结合CpG 1826的细胞占总细胞的百分比由9.7%减少为4%(P＜0.05),使摄取CpG 1826的细胞占总细胞的百分比由36%减少为13%(P＜0.01)。(5)半定量RT-PCR结果显示,体外培养时,CpG 1826刺激后小鼠脾细胞TLR9mRNA大量表达,ODN F546显著降低CpG 1826引起的TLR9 mRNA表达(P＜0.01)。【结论】Sj22.6抗原基因序列中存在具有明显免疫抑制作用的ODNF546和F311,它们特异性抑制刺激性CpG诱导的淋巴细胞增殖,抑制作用呈现一定的剂量效应关系和时间效应关系。ODN F546能抑制刺激性CpG诱导的细胞因子IFN-γ和IL-12的分泌。减少细胞对刺激性CpG的结合及摄取、降低刺激性CpG特异性受体TLR9的基因表达可能与其作用机制相关。研究结果提示存在免疫抑制性ODN可能与编码Sj22.6kDa抗原的核酸疫苗不能诱导产生有效保护力有关。
[Abstract]:[Objective] to study whether there are immunosuppressive fragments in the sequence of 22.6kDa (Sj22.6) antigen of Schistosoma japonicum.
[Methods] according to the structural features of the reported immunosuppressive sequences, the oligodeoxynucleotides (oligodeoxynucleotide, ODN), with a number of 20 to 30bp, were selected from the sequence of the gene encoding Sj22.6kDa antigen, and these short DNA sequences were synthesized artificially, with the internationally recognized ODN of strong immune stimulation to mice. CpG1826 was used as a stimulant in the follow-up experiment, and the control was set up. The spleen mononuclear cells of normal mice were isolated and cultured in vitro. The different ODN and CpG 1826 were added to the cell culture system. The proliferation of lymphocytes was observed by the isotope ~3H-TdR incorporation test, and the proliferation level of each group was observed in order to screen the inhibitory ODN. The relationship between the dose effect and the time effect of inhibitory ODN. Using A (Concanavalin A, ConA) as a stimulator. The effect of the inhibitory ODN on the proliferation of ConA induced cell proliferation was observed by ~3H-TdR incorporation assay. The inhibitory ODN was observed in vivo and in vitro by enzyme linked immunosorbent assay (ELISA) for CpG 1826, respectively. The effects of Th1 type cytokine IFN- gamma and IL-12 secretion were induced. Flow cytometry was used to detect the effects of inhibitory ODN on cell binding and uptake of CpG 1826. Finally, semi quantitative PCR was used to detect the effect of inhibitory ODN on the expression of Toll like receptor 9 (TLR9) mRNA expression of CpG 1826.
[results] (1) the lymphocyte proliferation test showed that ODN F546 and F311 in the Sj22.6kDa antigen gene sequence could inhibit the proliferation of spleen lymphocytes induced by CpG 1826 in vitro. The inhibition rates of ODN F546 and F311 were 87% and 11%. (2) lymphocyte proliferation test, respectively, when the ODN terminal concentration was 1 Mu M, respectively, when the amount of CpG 1826 was measured. The inhibitory effects of ODN F546 and F311 were enhanced as their amount increased. When the molar ratio of CpG 1826 was 1 to 1, the inhibitory effects of ODN F546 or F311 were strongest at the same time or before CpG 1826. (3) enzyme linked immunosorbent assay showed that in vitro and in vivo, ODN F546 significantly reduced the CpG 1826 induced mouse spleen The secretion of Th1 type cytokine IFN- gamma and IL-12 produced by the cells. (4) flow cytometry results showed that ODN F546 reduced the percentage of cells with CpG 1826 to total cells from 9.7% to 4% (P < 0.05), and reduced the percentage of CpG 1826 to total cells from 36% to 13% (P < 0.01). (5) semi quantitative RT-PCR results showed that body When cultured in vitro, CpG 1826 stimulated TLR9mRNA expression in splenocytes of mice, and ODN F546 significantly decreased TLR9 mRNA expression induced by CpG 1826 (P < 0.01).
[Conclusion] there is an obvious immunosuppressive effect of ODNF546 and F311 in the Sj22.6 antigen gene sequence. They specifically inhibit the proliferation of lymphocytes induced by stimulating CpG. The inhibitory effect presents a certain dose effect relationship and time effect relationship..ODN F546 can inhibit the secretion of IFN- gamma and IL-12 induced by stimulating CpG induced cytokines. The gene expression of CpG specific receptor TLR9 may be related to the binding and uptake of small cells to stimulating CpG, and the results suggest that the existence of immunosuppressive ODN may be related to the inability of the nucleic acid vaccine to encode the Sj22.6kDa antigen to induce effective protection.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392

【参考文献】