Studies on the immune response and antioxidant function of bacterial stimulation of the prx4 gene in crayfish (Procambarus clarkii)
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摘要:
目的 克隆克氏原螯虾（Procambarus clarkii）过氧化物还原酶4基因（Pc-prx4），分析Pc-prx4基因的正常组织表达以及细菌刺激后的相对表达情况，分析其抗氧化功能，为Pc-prx4在病原刺激后的先天免疫机制以及抗氧化功能研究提供参考依据。 方法 以Pc-prx4为目的基因，通过相关网站和软件（Expasy、Translate tool、SMART、Expasy ProtParam tool、BLASTx、MEGA-X）进行生物信息学分析。通过qRT-PCR检测Pc-prx4在正常组织中的表达量以及金黄色葡萄球菌（Staphylococcus aureus）、鲶爱德华氏菌（Edwardsiella ictaluri）刺激下的表达量；通过构建表达菌株、诱导纯化rPc-PRX4蛋白，对重组蛋白进行抗氧化功能研究。 结果 Pc-prx4开放阅读框744 bp，编码247个氨基酸，含有烷基氢过氧化物还原酶（AhpC）结构域、巯基特异性抗氧化结构域和1-Cys prx过氧铁氧酶的C末端结构域；Pc-PRX4蛋白分子式为 C1249H1934N330O357S10，分子量为27.61 kDa，理论等电点（pI）为5.88，N端存在信号肽区域，C端存在1-Cys过氧化物还原蛋白结构域。Pc-prx4基因在各组织中均有所表达，其中血淋巴的表达量最高；在金黄色葡萄球菌和鲶爱德华氏菌刺激下，Pc-prx4基因在血细胞、肝胰腺、鳃、肠组织中表达均呈现整体升高的趋势。在保护质粒DNA抗氧化缺刻试验中，rPc-PRX4表现出抗氧化性，并随着重组蛋白浓度的升高，抗氧化效果越明显。 结论 Pc-prx4基因属于1-Cys prx，在血细胞中高表达，参与调节金黄色葡萄球菌、鲶爱德华氏菌刺激下的机体免疫应答过程，rPc-PRX4蛋白还具有与浓度大小相关的抗氧化功能，该基因参与免疫过程的调节和保护机体进行抗氧化的功能。 Abstract: : [ Objective]To analyze the normal tissue expression of Pc-prx4 gene and its relative expression after bacterial stimulation by cloning Procambarus clarkii peroxiredoxin 4 gene, and the antioxidant function of Pc-prx4 gene, and to provide a reference basis for the study of innate immunity mechanism and the antioxidant function of Pc-prx4 gene in Pc-prx4 after pathogen stimulation. mechanism and the antioxidant function of this gene after pathogen stimulation.Methods Pc-prx4 was used as the target gene for the study. Bioinformatics analysis was carried out by Sangshin's website and software (Expasy, Translate tool, SMART, Expasy ProtParam tool, BLASTx, MEGA-X), The expression of Pc-prx4 in normal tissues and under the stimulation of Staphylococcus aureus and Edwardsiella ictaluri was detected by qRT-PCR; the recombinant protein was investigated for its antioxidant function by constructing the expression strains and inducing and purifying rPc-PRX4. Results The Pc-prx4 gene open reading frame is 744 bp, encoding 247 amino acid and contains an alkyl hydroperoxide reductase (AhpC) structural domain, a sulfhydryl-specific antioxidant structural domain, and the C-terminal structural domain of 1-Cys prx peroxisome iron oxidase. The Pc-PRX4 protein molecular formula is C1249H1934N330O357S10, molecular weight is 27.61 kDa, theoretical isoelectric point (pI) is 5.88, the signal peptide region exists at the N-terminal end, and the 1-Cys peroxiredoxin domain exists at the C-terminal end; the Pc-prx4 gene was expressed in all the sampled tissues, among which the expression of hemolymph was the highest.Pc-prx4 gene expression showed an overall elevated trend in hemocytes, hepatopancreas, gills, and intestinal tissues under the stimulation of Staphylococcus aureus and Edwardsiella catarrhalis..In the plasmid DNA protection assay, rPc-PRX4 exhibited antioxidant properties and tended to be obvious with the increase of recombinant protein concentration, Conclusion Pc-prx4 gene belongs to 1-Cys prx, is highly expressed in hemolymph and participates in the antioxidant homeostasis in the organism stimulated by S. aureus. rPc-PRX4 protein also has a concentration size-related The rPc-PRX4 protein also has a concentration size-related antioxidant function, and this gene has both an antioxidant function involved in the regulation of immune processes and protection of the organism. -
图 1 Pc-prx4目的基因扩增
M：标准DNA Marker；1～5：目的基因PCR扩增条带；6为阴性对照。
Figure 1. Results of Pc-prx4 gene amplification
M: Standard DNA Marker; 1～5: PCR amplified bands of target genes; 6: Negative control.
图 2 Pc-prx4序列功能结构域分析
红色区域为信号肽区域，Pfam Redoxin为氧化还原蛋白结构域，Pfam AhpC-TSA为烷基过氧化氢还原酶结构域，Pfam 1-Cys Prx-C为1-Cys过氧化物还原蛋白的C端结构域。
Figure 2. Functional domain analysis of Pc-prx4 sequence
The red region is the signal peptide region, Pfam Redoxin is the REDOX protein domain, Pfam AhpC-TSA is the alkyl peroxide reductase domain, and Pfam 1-Cys Prx-C is the C-terminal domain of 1-Cys peroxidase reductase.
图 3 Pc-Prx4的cDNA序列及对应氨基酸残基
黄色背景区域为信号肽区域，紫色字体为功能结构区域，绿色背景为起始密码子atg和终止密码子tag，红色背景区域为半胱氨酸（Cys），右侧数字分别对应核苷酸与氨基酸数目。
Figure 3. cDNA sequence and corresponding amino acid residues of Pc-Prx4
The yellow background area is the signal peptide region, the purple text is the functional structure region, the green background area is the start codon atg and tag stop codon, the red background area is cysteine (Cys), the numbers on the right correspond to the number of nucleotides and amino acid respectively.
图 4 Pc-Prx4氨基酸序列与其他物种氨基酸序列比对结果
Figure 4. Comparison of the amino acid sequence of Pc-Prx4 with that of other species
图 6 Pc-prx4正常组织分布的相对表达量
不同字母表示不同组织间差异显著（P＜0.05）
Figure 6. Relative expression of Pc-prx4 in normal tissue distribution
Different letters indicate significant differences among different tissues (P ＜ 0.05).
图 7 Pc-prx4金黄色葡萄球菌刺激下组织的相对表达量
A为血细胞，B为肝胰腺，C为鳃组织，D为肠组织。不同字母表示不同时间点间差异显著（P＜0.05）。图8同。
Figure 7. Relative expression of Pc-prx4 in tissues stimulated by Staphylococcus aureus
A: hemocytes; B: hepatopancreas; C: gills; D: intestines. Different letters indicate significant differences at different time (P＜0.05). Same for Table 8.
图 8 Pc-prx4鲶爱德华氏菌刺激下组织的相对表达量
Figure 8. Relative expression of Pc-prx4 in tissues stimulated by Edwardsiella ictaluri
图 9 rPc-PRX4重组蛋白电泳
M：标准蛋白Marker；1～4分别为诱导前、诱导后、破碎后上清、破碎后沉淀；5～8为纯化后的rPc-PRX4蛋白。
Figure 9. Electrophoretic diagram of rPc-PRX4 recombinant protein
M: Standard protein Marker; 1～4: before induction, after induction, after crushing supernatant, after crushing precipitation; 5～8: purified rPc-PRX4 protein.
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