鸡Caspase 3基因的克隆表达、酶活性及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2024.10.002

摘要/Abstract

摘要： 【目的】构建鸡半胱氨酸天冬氨酸蛋白酶3(chCaspase 3)基因表达载体，鉴定表达蛋白酶活性，并进行生物信息学分析，为后续研究chCaspase 3功能奠定基础。【方法】以鸡法氏囊组织cDNA为模板，通过PCR扩增chCaspase 3基因，构建chCaspase 3基因的原核和真核表达载体。将原核表达载体转化大肠杆菌BL21(DE3)感受态细胞，经IPTG诱导表达重组蛋白后利用Western blotting分析chCaspase 3原核表达产物。将真核表达载体以不同剂量(0、0.5、1.0、1.5、2.0 μg)转染DF-1细胞进行表达，利用Western blotting和间接免疫荧光技术(IFA)分析chCaspase 3真核表达产物。用Caspase 3活性检测试剂盒分别检测原核和真核表达蛋白的酶活性；利用生物信息学软件分析chCaspase 3的相似性，并预测chCaspase 3蛋白的结构域和三级结构。【结果】 chCaspase 3基因CDS区长852 bp,编码283个氨基酸。本研究成功构建了原核pET28a(+)-chCaspase 3和真核p3×Flag-CMV7.1-chCaspase 3重组质粒，原核表达产物分子质量为36 ku，与预期相符；真核表达产物表达量随转染的真核表达载体量的增加而增加。原核和真核表达产物均具有Caspase 3酶活性。生物信息学分析发现，chCaspase 3与其他物种相似性较低，且亲缘关系较远，chCaspase 3蛋白含有Caspase家族的保守五肽QACRG和经典的CASc结构域，其三维结构与人Caspase 3(hCaspase 3)十分吻合。【结论】试验成功克隆并表达了chCaspase 3基因，重组蛋白具有Caspase 3酶活性，生物信息学预测chCaspase 3蛋白与hCaspase 3蛋白有相似的三维结构，为揭示chCaspase 3蛋白的功能及探索其抗病毒天然免疫机制奠定了基础。

关键词: 鸡Caspase 3基因; 原核表达; 真核表达; 酶活性; 生物信息学

Abstract: 【Objective】 The purpose of this study was to construct expression vectors for chicken Caspase 3 (chCaspase 3) gene，identify the protease activity of the expressed protein，and conduct bioinformatics analysis，so as to lay a foundation for further research into the function of chCaspase 3. 【Method】 The cDNA from chicken bursa of Fabricius tissue was used as template for amplification of the chCaspase 3 gene via PCR. The recombinants of both prokaryotic and eukaryotic expression vectors for chCaspase 3 gene were then constructed. The recombinant prokaryotic expression vector was transformed into Escherichia coli BL21(DE3). Following IPTG induction，prokaryotic expression products of chCaspase 3 were subjected to Western blotting analysis. DF-1 cells were transfected with the eukaryotic expression vector in varying amounts(0，0.5，1.0，1.5 and 2.0 μg). The expression products were then analyzed using Western blotting and indirect immunofluorescence assay (IFA). Enzyme activity of the expressed proteins，both prokaryotic and eukaryotic，was assessed using Caspase 3 activity assay kits. Bioinformatic software was employed to assess the similarity of the chCaspase 3 and to forecast its protein’s structural domains and tertiary structure. 【Result】 The CDS region length of the chCaspase 3 gene was 852 bp，encoding 283 amino acids. Both prokaryotic pET28a(+)-chCaspase 3 and eukaryotic p3×Flag-CMV7.1-chCaspase 3 recombinant plasmids were constructed successfully. The prokaryotic expression product had a molecular weight of 36 ku，matching expectations. The expression of eukaryotic expression products were increased with the increase of transfected eukaryotic expression vectors. Caspase 3 enzyme activity was observed in both prokaryotic and eukaryotic expression products. Bioinformatics analysis showed the chCaspase 3 had low similarity with other species and distant phylogenetic relationships. The chCaspase 3 protein contained the conserved QACRG pentapeptide and CASc domain，characteristic of the Caspase family，mirroring the three-dimensional structure of human Caspase 3 (hCaspase 3). 【Conclusion】 The chCaspase 3 gene was cloned and expressed successfully，and its recombinant protein demonstrated Caspase 3 enzyme activity. Bioinformatics predictions revealed the three-dimensional structure of the chCaspase 3 protein closely resembled that of hCaspase 3. This similarity provided a basis for uncovering the function of chCaspase 3 and investigating its antiviral innate immunity.

Key words: chCaspase 3 gene; prokaryotic expression; eukaryotic expression; enzymatic activity; bioinformatics

中图分类号:

S831
Q78

陈扬, 项燕华, 李宜海, 何秀苗. 鸡Caspase 3基因的克隆表达、酶活性及生物信息学分析[J]. 中国畜牧兽医, 2024, 51(10): 4200-4210.

CHEN Yang, XIANG Yanhua, LI Yihai, HE Xiumiao. Cloning，Expression，Enzyme Activity and Bioinformatics Analysis of Caspase 3 Gene in Chicken[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(10): 4200-4210.

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