Immunogenecity of recombinant Leishmania donovani Peroxidoxin-1, tryparedoxin peroxidase, hypothetical protein CAJ07026.1 and GDP-mannose pyrophosphorylase in BALB/c mice

Abstract

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

CLC Number:

R382.22

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.

Figures/Tables 4

Table 1

Fig.1

Fig.2

Table 2

Cytokine expression fold-change in mouse spleen cells stimulated with recombinant rPxn1, rTryP, rHyp07026 and rGDPMP from Leishmania donovani in comparison to PBS group

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

Table 2

References 25

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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