细菌刺激下克氏原螯虾<i>prx4</i>基因的免疫响应和抗氧化功能研究

张明达; 李倩倩; 张文硕; 秦仕宇; 沈秀丽; 杜志强

doi:10.19303/j.issn.1008-0384.2023.10.007

细菌刺激下克氏原螯虾prx4基因的免疫响应和抗氧化功能研究

doi: 10.19303/j.issn.1008-0384.2023.10.007

1.
内蒙古科技大学生命科学与技术学院，内蒙古　包头 014010
2.
内蒙古科技大学图书馆，内蒙古　包头 014010

基金项目: 国家自然科学基金项目（32060834）；内蒙古科技大学2022年基本科研业务费专项（028）

详细信息

作者简介:
张明达（1999 —），男，硕士研究生，主要从事无脊椎动物先天免疫方向相关研究，E-mail：1501643202@qq.com

通讯作者:
杜志强（1980 —），男，博士，教授，主要从事无脊椎动物先天免疫方向相关研究，E-mail：nmdzq1981@163.com

中图分类号: S917.4
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出版历程
- 收稿日期: 2023-07-17
- 修回日期: 2023-10-07
- 网络出版日期: 2023-11-20
- 刊出日期: 2023-10-28

Immune Response and Antioxidant Activities of Bacteria-stimulated Prx4 in Procambarus clarkii

1.
College of Life Science and Technology, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia　014010, China
2.
Library, Inner Mongolia University of Science and Technology, Baotou, Inner Mongolia　014010, China

摘要

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。
- 克氏原螯虾 /
- 过氧化物还原酶4 /
- 金黄色葡萄球菌；鲶爱德华氏菌 /
- 表达模式 /
- 抗氧化
Abstract: Objective Immune response of peroxiredoxin 4 gene of Procambarus clarki (Pc-prx4) after being stimulated by bacterial infection was studied. Methods Pc-prx4 were cloned before and after an artificial inoculation of Staphylococcus aureus or Edwardsiella ictaluri on the crayfish. Bioinformatic analysis was conducted with the aid of relevant online websites and software including Expasy, translate tool, SMART, Expasy ProtParam tool, BLASTx, and MEGA-X. Expressions of Pc-prx4 from the tissues of normal and pathogenically inoculated crayfish were detected by qRT-PCR. Antioxidant function of the constructed recombinant protein was determined. Results Pc-prx4 had an open reading frame of 744 bp encoding 247 amino acids with an alkyl hydroperoxide reductase (AhpC) structural domain, a sulfhydryl-specific antioxidant structural domain, and a C-terminal structural domain of 1-Cys prx peroxisome iron oxidase. The predicted protein molecular formula was C₁₂₄₉H₁₉₃₄N₃₃₀O₃₅₇S₁₀ with a molecular weight of 27.61 kDa and a pI of 5.88. Its signal peptide region was located at the N-terminal end, and the 1-Cys peroxiredoxin domain at the C-terminal end. The gene expressed in all sampled tissues—most highly in the hemolymph and high in the hemocytes, hepatopancreas, gills, and intestines of the crayfish inoculated by S. aureus or E. catarrhalis. In the plasmid DNA protection assay, rPc-PRX4 displayed varying degrees of antioxidant activity, especially in high concentrations. Conclusion A kind of 1-Cys prx, Pc-prx4 was highly expressed in the hemolymph of P. clarkii stimulated by S. aureus or E. catarrhalis. The protein exhibited a concentration-dependent activity associated with the antioxidant homeostasis of the crayfish.
- Procambarus clarkii /
- peroxiredoxin 4 /
- Staphylococcus aureus; Edwardsiella ictaluri /
- expression of target genes /
- antioxidant

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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 amplification

M: Standard DNA marker; 1-5: PCR amplified bands of target genes; 6: negative control.

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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 domains and physicochemical properties of Pc-prx4

Red: signal peptide region; Pfam Redoxin: REDOX protein domain; Pfam AhpC-TSA: alkyl peroxide reductase domain; and Pfam 1-cys Prx_-C: C-terminal domain of 1-Cys peroxidase reductase.

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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 sequences and corresponding amino acid residues of Pc-Prx4

Yellow background: signal peptide region; purple text: functional structure region; green background: start codon atg and tag stop codon; red background: cysteine (Cys); and numbers: counts of nucleotides and amino acids, respectively.

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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. Amino acid sequences of Pc-Prx4 and other species

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图 5 Pc-Prx4与其他物种共建系统进化树

Figure 5. Co-constructed phylogenetic tree of Pc-Prx4 with other species

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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 expressions of Pc-prx4 in tissues of normal crayfish

Data with different letters indicate significant differences among tissues at P＜0.05.

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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 expressions of Pc-prx4 in tissues of crayfish stimulated by S. aureus

A: hemocytes；B: hepatopancreas；C: gills；D: intestines. Data with different letters indicate significant differences among different treatment time at P＜0.05. Same for Table 8.

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图 8 Pc-prx4鲶爱德华氏菌刺激下组织的相对表达量

Figure 8. Relative expressions of Pc-prx4 in tissues of crayfish stimulated by E. ictaluri

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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

M: Standard protein marker; 1–4: before induction, after induction, after crushing supernatant, and after crushing precipitation, respectively; 5–8: purified rPc-PRX4 protein.

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图 10 rPc-PRX4重组蛋白在金属催化氧化中对质粒DNA断裂的保护作用

Figure 10. Protection of rPc-PRX4 against plasmid DNA breakage in metal-catalyzed oxidation

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