重组杜氏利什曼原虫Pxn1、TryP、假定蛋白CAJ07026和GDPMP蛋白刺激BALB/c小鼠的免疫应答状态

摘要/Abstract

摘要：

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（M_r）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。

关键词: 杜氏利什曼原虫, 过氧化物还原酶1, 锥虫氧化还原过氧化物酶, 假定蛋白CAJ07026, 甘露糖-1-磷酸胍氨酸转移酶, 免疫原性

Abstract:

Objective To evaluate the immune responses to recombinant Leishmania donovani peroxidoxin-1(Pxn1), tryparedoxin peroxidases(TryP), hypothetical protein CAJ07026.1(Hyp07026) and GDP-mannose pyrophosphorylase(GDPMP) in BALB/c mice.Methods The genes of L.donovani Pxn1, TryP, Hyp07026 and GDPMP were amplified by RT-PCR separately, and cloned into plasmid pQE30 to construct recombinant pQE30-Pxn1, pQE30-TryP, pQE30-Hyp07026 and pQE30-GDPMP, respectively.They were then transfected into Escherichia coli for expression under IPTG induction.rPxn1 and rTryP were purified with the NTA-Ni purification system under native condition, while rHyp07026 and rGDPMP were purified with the same system under denaturing condition.Then the recombinant proteins were used to immunize BALB/c mice (n = 5 for each) for 4 times and PBS was used as a control (n = 5).After that, RNA was extracted from spleen cells, and the transcriptional profiling of cytokines was examined by qRT-PCR microarray.Results PCR amplification of Pxn1, TryP, Hyp07026 and GDPMP produced fragments of 570, 610, 1 040 and 1 200 bp, respectively, sequences of which were consistent with those in GenBank as confirmed by the sequencing results.All of them were highly expressed in E.coli under IPTG induction, with molecular weights of 22 000, 23 000, 38 000 and 45 000 for rPXNT1, rTXNT, rHyp07026 and rGDPMP, respectively.Among the mRNAs of 84 cytokine genes expressed in the spleen cells of the BALB/c mice immunized with rGDPMP, rPXNT1, rTXNT and rHyp07026, significantly higher mRNA levels of IL-1f6( 96.22, 271.54, 108.51 and 250.15, respectively ), IL-17f (105.54, 35.71, 53.38 and 53.57, respectively ) and IL-10 (21.68, 27.51, 24.45 and 2.91, respectively ) were found in comparison to PBS control, with significantly lower mRNA levels of IL-4 (5.74, 2.43, 1.59 and 0.76, respectively), IL-12β (0.05, 0.26, 0.10 and 0.50, respectively), IL-18 (0.12, 0.21, 0.13 and 0.04, respectively ) and IFN-γ(3.59, 0.39, 0.29 and 0.63, respectively).Conclusion The findings suggest that the rTryP, rPxn1, rHyp07026 and rGDPMP of L.donovani can stimulate inflammatory responses and induce immune suppression.

Key words: Leishmania donovani, Peroxidoxin-1, Tryparedoxin peroxidase, Hypothetical protein CAJ07026.1, GDP-mannose pyrophosphorylase, Immunogenecity

中图分类号:

R382.22

敬保迁, 谢勇恩, 胡为民, 杨健, 王朝莉, 冯莉. 重组杜氏利什曼原虫Pxn1、TryP、假定蛋白CAJ07026和GDPMP蛋白刺激BALB/c小鼠的免疫应答状态[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(6): 563-569.

Bao-qian JING, Yong-en XIE, Wei-min HU, Jian YANG, Chao-li WANG, Li FENG. Immunogenecity of recombinant Leishmania donovani Peroxidoxin-1, tryparedoxin peroxidase, hypothetical protein CAJ07026.1 and GDP-mannose pyrophosphorylase in BALB/c mice[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(6): 563-569.

图/表 4

表1

图1

图2

表2

重组杜氏利什曼原虫rPxn1、rTryP、rHyp07026和rGDPMP 免疫小鼠后其脾细胞细胞因子表达与对照组相比的变化倍数

基因标志 Gene symbol	重组过氧化物还原酶1 rPxn1		重组锥虫氧化还原过氧化物酶rTryP		重组假定蛋白 rHyp07026		重组GDP-甘露糖焦磷酸化酶rGDPMP
基因标志 Gene symbol	变化倍数 Fold-change	P值 P value	变化倍数 Fold-change	P值 P value	变化倍数 Fold-change	P值 P value	变化倍数 Fold-change	P值 P value
Bmp1	1.97	0.037 5	1.23	0.569 2	0.75	0.442 0	0.59	0.509 0
Bmp10	5.21	0.068 9	3.01	0.090 7	8.34	0.001 5	2.45	0.179 2
Bmp2	0.87	0.600 5	0.15	0.015 0	0.28	0.016 2	0.16	0.076 6
Bmp3	1.10	0.254 0	1.46	0.003 5	1.50	0.002 2	0.67	0.001 6
Bmp4	0.39	0.115 6	0.25	0.005 4	0.06	0.000 4	0.28	0.005 2
Bmp5	1.10	0.254 0	1.46	0.003 5	1.50	0.002 2	0.67	0.001 6
Bmp6	0.56	0.065 5	0.42	0.008 6	0.04	0.006 0	0.21	0.003 5
Bmp7	0.60	0.416 6	0.42	0.018 3	0.07	0.000 0	0.45	0.006 1
Bmp8b	1.90	0.119 0	0.60	0.320 0	0.91	0.830 9	0.31	0.067 8
Csf1	3.01	0.000 4	1.93	0.002 6	1.44	0.020 6	1.84	0.022 9
Csf2	2.06	0.014 4	1.63	0.273 8	4.27	0.004 0	6.55	0.001 2
Ctf1	8.85	0.018 5	1.28	0.734 8	2.98	0.244 2	3.13	0.106 4
Ctf2	1.10	0.254 0	1.46	0.003 5	1.50	0.002 2	0.67	0.001 8
Fasl	0.67	0.052 6	0.53	0.019 0	3.40	0.002 0	0.61	0.237 4
Fbrs	1.15	0.026 6	0.97	0.763 1	1.30	0.026 4	0.70	0.003 7
Fgf10	1.10	0.254 0	1.46	0.003 5	2.53	0.054 1	0.67	0.001 6
Flt3l	0.59	0.000 2	0.60	0.000 0	0.41	0.216 8	0.52	0.000 0
Gdf1	0.85	0.440 9	1.08	0.610 3	1.38	0.148 2	0.45	0.010 1
Gdf10	0.31	0.077 3	0.17	0.000 4	0.26	0.030 0	0.08	0.000 1
Gdf11	0.21	0.000 0	0.22	0.000 3	0.01	0.000 1	0.15	0.000 1
Gdf15	18.79	0.000 0	12.28	0.000 2	14.96	0.000 1	2.47	0.015 4
Gdf2	0.96	0.900 2	1.03	0.923 6	0.98	0.952 9	0.65	0.361 8
Gdf3	0.99	0.960 6	0.23	0.073 9	0.81	0.547 4	0.58	0.131 5
Gdf5	2.15	0.175 9	3.99	0.014 7	1.91	0.184 5	0.95	0.926 7
Mstn	1.10	0.254 0	1.46	0.003 5	1.50	0.002 2	0.67	0.001 6
Gdf9	0.77	0.375 3	0.78	0.623 9	0.17	0.000 2c	0.51	0.185 5
Ifna2	1.47	0.622 5	1.74	0.276 0	1.21	0.546 0	0.96	0.932 1
Ifna4	3.34	0.195 6	1.87	0.408 5	0.80	0.702 2	1.29	0.725 3
Ifnb1	1.10	0.254 0	1.46	0.003 5	1.50	0.002 2c	0.84	0.117 5
Ifng	0.39	0.000 2	0.29	0.000 8	0.63	0.007 8c	3.59	0.000 1
Il10	27.51	0.000 0	24.45	0.000 0	2.91	0.007 9c	21.68	0.000 0
Il11	1.64	0.167 9	1.42	0.024 2	1.55	0.055 1	0.93	0.827 6
Il12b	0.26	0.014 1	0.10	0.037 7	0.50	0.583 8	0.05	0.002 2
Il13	2.23	0.015 5	1.60	0.087 7	2.10	0.004 8	5.84	0.000 3
Il15	0.97	0.857 8	0.78	0.161 1	0.09	0.000 1	0.76	0.107 8
Il16	0.38	0.019 3	0.30	0.012 7	0.50	0.054 8	0.19	0.007 4
Il17b	1.03	0.976 4	1.06	0.923 8	1.81	0.413 6	0.55	0.336 5
Il17c	1.65	0.331 5	0.54	0.256 5	1.03	0.975 5	0.37	0.122 0
Il25	1.10	0.254 0	1.46	0.003 5	1.50	0.002 2	0.67	0.001 6
Il17f	35.71	0.000 1	53.38	0.000 0	53.57	0.000 0	105.54	0.000 0
Il18	0.21	0.000 0	0.13	0.000 2	0.04	0.002 9	0.12	0.000 0
Il19	15.33	0.000 1	7.00	0.012 8	2.17	0.109 7	3.45	0.000 7
Il1a	16.02	0.000 1	9.46	0.000 3	4.77	0.001 4	20.18	0.000 1
Il1b	9.44	0.000 0	4.50	0.000 2	3.66	0.001 5	2.56	0.002 4
Il1f10	3.31	0.112 4	4.00	0.059 8	1.50	0.002 2	1.90	0.116 6
Il1f5	3.30	0.162 5	2.92	0.081 9	1.23	0.365 5	1.17	0.853 2
基因标志 Gene symbol	重组过氧化物还原酶1 rPxn1		重组锥虫氧化还原过氧化物酶rTryP		重组假定蛋白Hyp07026rHyp07026		重组GDP-甘露糖焦磷酸化酶rGDPMP
基因标志 Gene symbol	变化倍数 Fold-change	P值 P value	变化倍数 Fold-change	P值 P value	变化倍数 Fold-change	P值 P value	变化倍数 Fold-change	P值 P value
Il1f6	271.54	0.000 0	108.51	0.000 0	250.15	0.000 0	96.22	0.000 0
Il1f8	2.62	0.129 9	1.72	0.020 4	2.32	0.113 2	0.67	0.001 6
Il1f9	19.45	0.000 0	11.33	0.000 0	48.73	0.000 0	6.11	0.000 1
Il1rn	47.01	0.000 0	37.40	0.000 0	24.03	0.000 1	17.77	0.000 1
Il2	6.60	0.016 1	3.27	0.0813	5.23	0.036 7	2.52	0.197 0
Il20	1.10	0.254 0	1.46	0.003 5	1.50	0.002 2	0.90	0.727 6
Il21	3.34	0.008 8	1.55	0.237 0	13.00	0.000 5	3.58	0.016 9
Il24	0.15	0.000 0	0.16	0.000 1	0.01	0.000 0	0.11	0.000 0
Il27	3.12	0.000 6	2.69	0.002 9	8.36	0.000 1	2.69	0.000 7
Il3	41.02	0.000 0	14.27	0.000 6	5.79	0.005 9	83.96	0.000 0
Il4	2.43	0.016 9	1.59	0.197 1	0.71	0.379 8	5.74	0.000 6
Il7	1.10	0.254 0	1.46	0.003 5	2.38	0.138 4	0.67	0.001 6
Il9	1.10	0.254 0	1.46	0.003 5	1.50	0.002 2c	0.84	0.506 2
Inha	2.32	0.086 2	3.32	0.045 1	0.43	0.200 9	4.76	0.015 9
Inhba	30.95	0.000 1	20.46	0.000 1	35.51	0.000 1	24.00	0.000 2
Lefty1	0.50	0.004 8	0.51	0.016 9	0.32	0.000 5	0.36	0.002 1
Lif	51.33	0.000 3	33.09	0.000 5	10.65	0.004 4	46.05	0.000 6
Lta	0.44	0.000 3	0.24	0.002 3	0.59	0.004 6	0.58	0.004 3
Ltb	0.18	0.000 0	0.14	0.000 0	0.26	0.000 0	0.14	0.000 0
Mif	1.93	0.002 1	1.92	0.003 1	3.65	0.000 2	1.98	0.003 2
Scgb3a1	11.22	0.0108	6.89	0.021 7	5.66	0.057 5	3.19	0.113 3
Scye1	0.99	0.922 4	1.05	0.661 3	1.84	0.009 6	1.02	0.593 3
Tnf	3.58	0.031 6	1.07	0.878 5	1.69	0.246 2	0.87	0.847 0
Tnfrsf11b	9.09	0.003 9	2.30	0.288 8	2.54	0.103 9	6.78	0.016 0
Tnfsf10	0.74	0.000 9	0.74	0.024 3	0.75	0.004 7	0.72	0.000 1
Tnfsf11	0.88	0.638 9	0.66	0.105 0	0.18	0.002 5	0.71	0.264 2
Tnfsf12	1.32	0.008 7	0.76	0.046 4	1.39	0.0340	1.17	0.049 6
Tnfsf13	0.51	0.017 5	0.24	0.001 4	2.69	0.004 6	0.35	0.008 7
Tnfsf13b	0.71	0.005 2	0.57	0.003 1	0.32	0.002 3	0.44	0.012 8
Tnfsf14	0.92	0.457 2	0.65	0.016 0	0.74	0.065 3	0.65	0.056 8
Tnfsf15	2.87	0.260 5	1.53	0.631 9	1.43	0.642 8	1.21	0.807 2
Tnfsf18	1.60	0.145 2	1.52	0.355 5	0.56	0.110 7	0.96	0.882 3
Tnfsf4	0.53	0.026 5	0.46	0.056 8	0.26	0.004 0	0.50	0.001 7
Cd40lg	0.56	0.019 9	0.45	0.007 9	0.40	0.006 4	0.39	0.000 8
Cd70	14.37	0.000 0	9.77	0.000 1	0.34	0.000 7	9.20	0.000 2
Tnfsf8	2.28	0.017 4	1.44	0.169 9	2.07	0.043 7	2.07	0.039 8
Tnfsf9	5.46	0.009 2	4.15	0.013 3	3.03	0.033 7	1.95	0.225 4
Txlna	0.90	0.798 3	0.69	0.403 9	0.80	0.599 9	0.39	0.159 0
Gusb	1.66	0.058 2	1.10	0.654 8	2.83	0.010 3	1.21	0.572 7
Hprt1	1.06	0.250 5	1.28	0.001 3	0.82	0.0162	0.99	0.820 4
Hsp90ab1	0.94	0.250 5	0.78	0.001 3	1.22	0.016 2	1.01	0.820 4
Gapdh	1.18	0.009 0	1.00	0.799 4	1.72	0.000 2	1.62	0.000 0
Actb	0.74	0.061 2	0.59	0.009 8	0.84	0.239 0	0.53	0.004 3
Bmp1	1.97	0.061 2	1.23	0.061 1	0.75	0.009 8	0.59	0.004 3

表2

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基因名称 Gene name	引物序列5′→3′ Primer sequences5′→3′	酶切位点 Restriction site
Hyp07026	F： CGGATCCTACGCGGGCTTTCCGAGG	BamHⅠ
Hyp07026	R： GAAGCTTATTACAGGTCGCGCAG- CAACGAAG	HindⅢ
Pxn1	F： GGGATCCTCCTGCGGTGACGCCAAG	BamHⅠ
Pxn1	R： CAAGCTTATTACTTATTGTGATC- GACCTTCAGGCC	HindⅢ
TryP	F： GGG ATCCTCCTGCGGTAACGCCAAG	BamHⅠ
TryP	R： CAAGCTTATTACTGCTTGCTGAAG-TATCCCTCGAC	HindⅢ
GDPMP	F： GGGATGGTCTTCATCCGATGGCCAGG	BamHⅠ
GDPMP	R： CTTATTACATGATGATACCAGG- CTCGAAGTG