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目的 观察重组杜氏利什曼原虫（Leishmania donovani）过氧化物还原酶1（peroxidoxin-1，Pxn1）、锥虫氧化还原过氧化物酶（tryparedoxin peroxidase，TryP）、假定蛋白CAJ07026（hypothetical protein CAJ07026.1，Hyp07026）和甘露糖-1-磷酸胍氨酸转移酶（GDP-mannose pyrophosphorylase，GDPMP）蛋白刺激BALB/c小鼠免疫应答状况。 方法 逆转录PCR（RT-PCR）分别扩增杜氏利什曼原虫Pxn1、TryP、Hyp07026和GDPMP编码基因，克隆入质粒pQE30，构建重组质粒pQE30-Pxn1、pQE30-TryP、pQE30-Hyp07026和pQE30-GDPMP，转化至大肠埃希菌，以异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达。采用NTA-Ni盐凝胶层析柱，自然条件方式纯化重组蛋白rPxn1和rTryP，变性条件方式纯化重组蛋白rHyp07026和rGDPMP。25只BALB/c小鼠随机均分成5组，即rPxn1、rTryP、rHyp07026、rGDPMP免疫组及PBS对照组，免疫组小鼠分别以相应重组蛋白进行免疫，第1周免疫组小鼠皮下注射100 μg相应的重组蛋白，隔周以50 μg相应的重组蛋白加强免疫3次，对照组小鼠注射等量PBS。第3次加强免疫后1周，将各组小鼠脱颈处死，制备单个脾细胞，提取抗原刺激后小鼠脾细胞总RNA, 用qRT-PCR微阵列检测抗原刺激后小鼠脾细胞内反映免疫应答的细胞因子转录水平表达谱。 结果 Pxn1、TryP、Hyp07026和GDPMP编码基因经PCR扩增，其产物大小分别为570、610、1 040和1 200 bp；经测序证实，与GenBank中的相关序列一致。重组质粒pQE30-Pxn1、pQE30-TryP、pQE30-Hyp07026和pQE30-GDPMP经IPTG诱导后均可在大肠埃希菌内高效表达，rGDPMP、rPXNT1、rTXNT和rHyp07026蛋白相对分子质量（Mr）分别为45 000、22 000、23 000和38 000。rGDPMP、rPXNT1、rTXNT和rHyp07026免疫小鼠的脾细胞内84个细胞因子基因的mRNA表达中，与PBS对照小鼠脾细胞比较，高水平表达的IL-1f6分别是对照组的96.22、271.54、108.51和250.15倍，IL-17f分别是对照组的105.54、35.71、53.38和53.57倍，IL-10分别是对照组的21.68、27.51、24.45和2.91倍。低水平表达的IL-4分别是对照组的5.74、2.43、1.59和0.71倍，IL-12β分别是对照组的0.05、0.26、0.10和0.50倍，IL-18分别是对照组的0.12、0.21、0.13和0.04倍，IFN-γ分别是对照组的3.59、0.39、0.29和0.63倍。 结论 杜氏利什曼原虫重组蛋白rPxn1、rTryP、rHyp07026和rGDPMP可刺激小鼠炎症反应及诱导免疫抑制。
Objective To observe the effects of recombinant Leishmania donovani peroxidoxin-1 (Pxn1), Tryparedoxin peroxidase (TryP), hypothetical protein CAJ07026.1 , Hyp07026) and GDP-mannose pyrophosphorylase (GDPMP) protein in BALB / c mice. Methods The Leishmania donovani Pxn1, TryP, Hyp07026 and GDPMP genes were amplified by RT-PCR and cloned into plasmid pQE30 to construct recombinant plasmids pQE30-Pxn1, pQE30-TryP, pQE30-Hyp07026 and pQE30-GDPMP , Transformed into Escherichia coli, and induced with isopropyl-β-D-thiogalactoside (IPTG). Recombinant proteins rPxn1 and rTryP were purified by natural conditions using NTA-Ni salt gel chromatography column. The recombinant proteins rHyp07026 and rGDPMP were purified by denaturing conditions. Twenty five BALB / c mice were randomly divided into five groups: rPxn1, rTryP, rHyp07026, rGDPMP and PBS control groups. The immunized mice were immunized with corresponding recombinant proteins respectively. The first week of immunization mice were subcutaneously injected with 100 μg of corresponding recombinant protein, immunized three times with 50 μg of the corresponding recombinant protein every other week, and the control group mice were injected with the same amount of PBS. One week after the third booster immunization, the mice in each group were sacrificed and their spleen cells were prepared. Single spleen cells were prepared and total RNA was extracted from the spleen cells of the mice stimulated with the antigen. The spleen cells in the mice were stimulated with qRT-PCR microarray Cytokine transcriptional profiling of immune responses. Results The products of Pxn1, TryP, Hyp07026 and GDPMP were amplified by PCR and their sizes were 570, 610, 1 040 and 1 200 bp, respectively. The sequences of Pxn1, Tryp, Hyp07026 and GDPMP were consistent with those of GenBank. The recombinant plasmids pQE30-Pxn1, pQE30-TryP, pQE30-Hyp07026 and pQE30-GDPMP were all highly expressed in E. coli after induced by IPTG. The relative molecular weights of rGDPMP, rPXNT1, rTXNT and rHyp07026 were 45 000 , 22 000, 23 000 and 38 000. In spleen cells of mice immunized with rGDPMP, rPXNT1, rTXNT and rHyp07026, compared with the spleen cells of PBS control mice, the levels of IL-1f6 in high splenocytes were 96.22,271.54,108.51 And 250.15-fold, IL-17f respectively 105.54,35.71,53.38 and 53.57 times of the control group, IL-10 respectively 21.68,27.51,24.45 and 2.91 times of the control group. IL-4 expressed at low level were 5.74,2.43,1.59 and 0.71 times of that of control group, IL-12β was 0.05,0.26,0.10 and 0.50 times of control group, IL-18 was 0.12,0.21 of control group respectively, 0.13 and 0.04 times, IFN-γ were 3.59,0.39,0.29 and 0.63 times of the control group respectively. Conclusion Leishmania donovani recombinant proteins rPxn1, rTryP, rHyp07026 and rGDPMP can stimulate inflammation and induce immunosuppression in mice.