| 斑马鱼g型溶菌酶基因序列分析及其原核表达 |
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| 引用本文： | 陈华,林晨韬,陈曦,葛均青.斑马鱼g型溶菌酶基因序列分析及其原核表达[J].南方农业学报,2022,53(1):229-237. |
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| 作者姓名： | 陈华 林晨韬 陈曦 葛均青 |
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| 作者单位： | 1 福建省农业科学院生物技术研究所, 福州 350003;2 福建师范大学生命科学学院/南方海洋研究院福建省微藻种质改良工程技术研究中心/福建省特色海洋生物资源可持续利用重点实验室, 福州 350117 |
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| 基金项目： | 福建省农业科学院自由探索科技创新项目;福建省属公益类科研院所基本科研专项;福建省农业科学院对外合作项目;福建省农业科学院协同创新工程建设项目 |
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| 摘 要： | 【目的】实现斑马鱼g型溶菌酶在大肠杆菌中表达，以获得高纯度且具溶菌活性的融合蛋白，为探究g型溶菌酶在斑马鱼抗菌过程中的作用机制及其开发利用打下基础。【方法】通过ClustalW、ExPASy、SignalP-5.0 Server及PSIPRED等在线软件对斑马鱼g型溶菌酶进行生物信息分析，经密码子优化后合成斑马鱼g型溶菌酶基因，亚克隆至pET-28a（+）表达载体并转化大肠杆菌BL21（DE3）感受态细胞，以IPTG进行诱导表达，并通过非干扰型蛋白浓度测定试剂盒和溶菌酶测定试剂盒（比浊法）测定其浓度及溶菌活性。【结果】从GenBank检索获得的斑马鱼溶菌酶基因g1（NM_001002706.1）和g2（XM_002664371.5）分别命名为Zeb-Lys-g1和Zeb-Lys-g2。Zeb-Lys-g1基因开放阅读框（ORF）长591 bp，共编码196个氨基酸残基，其编码蛋白分子量约21.6 kD；Zeb-Lys-g2的ORF长576 bp，共编码191个氨基酸残基，其编码蛋白分子量约21.1 kD；2种斑马鱼g型溶菌酶序列中均含有2个半胱氨酸残基及3个保守的催化残基位点（Glu、Asp和Asp）。Zeb-Lys-g1的N端含有17个氨基酸的信号肽，而Zeb-Lys-g2不存在典型的信号肽结构，但其三级结构均具有多个α-螺旋结构。在基于溶菌酶序列相似性构建的系统发育进化树中，Zeb-Lys-g1序列与金鱼g型溶菌酶序列的亲缘关系最近，而Zeb-Lys-g2序列与鲈形目和鲽形目鱼类的g型溶菌酶序列亲缘关系较近。将合成的斑马鱼g型溶菌酶基因（Zeb-Lys-g1和Zeb-Lys-g2）亚克隆至pET-28a（+）表达载体并转化BL21（DE3）感受态细胞，20 ℃下经IPTG（终浓度0.5 mmol/L）诱导16 h，可获得融合蛋白Zeb-Lys-g1和Zeb-Lys-g2，纯化后的浓度分别为1.01和1.66 mg/mL，对应的溶菌活性分别为689.68和44.39 U/mg。【结论】鱼类g型溶菌酶的基因变异较保守，经密码子优化及全基因合成方式合成的斑马鱼g型溶菌酶基因Zeb-Lys-g1和Zeb-Lys-g2可通过原核表达获得高纯度、高溶菌活性的融合蛋白，为探究斑马鱼溶菌酶的抗菌机制提供了技术支持。
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| 关 键 词： | 斑马鱼 g型溶菌酶 序列特征 原核表达 溶菌活性 |
| 收稿时间： | 2021-06-22 |
Sequence analysis and prokaryotic expression of zebrafish g-type lysozyme gene |
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| CHEN Hua,LIN Chen-tao,CHEN Xi,GE Jun-qing.Sequence analysis and prokaryotic expression of zebrafish g-type lysozyme gene[J].Journal of Southern Agriculture,2022,53(1):229-237. |
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| Authors: | CHEN Hua LIN Chen-tao CHEN Xi GE Jun-qing |
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| Affiliation: | 1 Institute of Biotechnology, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China;2 College of Life Sciences, Fujian Normal University/Center of Engineering Technology Research for Microalgae Germplasm Improvement of Fujian, Southern Institute of Oceanography/Fujian Key Laboratory of Special Marine Bio-resources Sustainable Utilization, Fuzhou 350117, China |
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| Abstract: | 【Objective】Zebrafish g-type lysozyme was expressed in Escherichia coli to obtain a fusion protein with high purity and bacteriolytic activity，which laid a foundation for the further study of antibacterial function and application of zebrafish g-type lysozyme.【Method】The biological information of g-type lysozyme gene of zebrafish was analyzed by online softwares such as ClustalW，ExPASy，SignalP-5.0 Server and PSIPRED. After coding codon optimization，g-type lysozyme gene of zebrafish was synthesized，respectively，and cloned into expression vector pET-28a（+）. The constructed plasmids were transformed into competent cells of E. coli BL21（DE3）. Then induced with IPTG to express the g-type lysozyme，whichwas used to determine its concentration and bacteriolytic activity by non-interference protein concentration determination kit and lysozyme determination kit（turbidimetry）.【Result】Zebrafish lysozyme genes g1（NM_001002706.1）and g2（XM_002664371.5）retrieved from GenBank were named Zeb-lys-g1 and Zeb-lys-g2，respectively. The open reading frame（ORF）of Zeb-lys-g1 gene was 591 bp in length and encoded 196 amino acid residues， with predicted molecular weight of 21.6 kD. The ORF of Zeb-lys-g2 was 576 bp in length and encoded 191 amino acid residues，with predicted molecular weight of 21.1 kD. The two zebrafish g-type lysozyme genes both had two cysteine residues and three conserved catalytic residues（Glu，Asp and Asp）. Zeb-lys-g1 contained 17 amino acid signal peptide at Nterminal，while Zeb-lys-g2 had no signal peptide，but its tertiary structure had multiple α-Spiral structure. In the phylogenetic tree constructed based on the similarity of lysozyme sequence，Zeb-lys-g1sequence was the closest to the g-type lysozyme sequence of goldfish，while Zeb-lys-g2 sequence was closer to the g-type lysozyme sequence of perch and flounder. Zebrafish g-type lysozyme genes（Zeb-lys-g1and Zeb-lys-g2）were subcloned intoexpression vector pET-28a（+）and transferred into competent cells of BL21（DE3）. The fusion proteins Zeb-lys-g1and Zeb-lys-g2 were obtained after induction by IPTG（final concentration 0.5 mmol/L）at 20 ℃ for 16 h. The purified concentrations were 1.01 and 1.66 mg/mL respectively，and the corresponding lysozyme activities were 689.68 and 44.39 U/mg，respectively.【Conclusion】The gene variation of fish g-lysozyme is relatively conservative. Zebrafish g-lysozyme genes zeb-lys-g1 and zeb-lys-g2 are synthesized by codon optimization and whole gene synthesis ，which cab obtain fusion proteins with high purity and high bacteriolytic activity through prokaryotic expression，which provides technical support for exploring the antibacterial mechanism of zebrafish lysozyme. |
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