单增李斯特菌IspD蛋白生物信息学分析及表达纯化

doi:10.16431/j.cnki.1671-7236.2023.09.036

摘要/Abstract

摘要： 【目的】对单增李斯特菌IspD蛋白的潜在生物学功能进行预测分析,并表达纯化IspD蛋白,为后续IspD蛋白的研究提供理论支撑。【方法】从NCBI中获得单增李斯特菌IspD的编码序列(基因ID:986270)和氨基酸序列(登录号:NP_464611.1),运用生物信息学软件对IspD蛋白编码序列进行开放阅读框分析,对氨基酸序列进行基本特性、空间结构、抗原表位、修饰化位点、蛋白互作及相似性等预测分析。对IspD蛋白编码序列进行密码子优化并合成,采用无缝克隆法构建重组质粒pET-32a-IspD并转化大肠杆菌BL21(DE3)感受态细胞,将测序和双酶切验证正确的阳性克隆经IPTG低温过夜诱导表达,采用镍柱亲和层析法纯化得到IspD融合蛋白,利用SDS-PAGE和Western blotting验证IspD融合蛋白表达、纯化及反应原性。【结果】IspD蛋白编码基因为lmo1086,全长711 bp,共有4个开放阅读框,其中ORF1为最长的开放阅读框,可编码236个氨基酸。氨基酸序列分析显示,IspD蛋白无跨膜结构域、信号肽,是一种亲水性蛋白,亚细胞定位于细胞质中,为2-C-甲基-D-赤藓糖醇-4-磷酸胞苷酰基转移酶。α-螺旋、延伸链、β-转角和无规则卷曲参与了IspD蛋白二级结构的组成,三级结构模型预测与二级结构特点相符。IspD蛋白具有多个T、B细胞抗原表位、固定无序结构域、磷酸化、糖基化、甲基化及乙酰化修饰位点。蛋白互作分析显示,IspD蛋白与DXR、IspF、ipk等多个蛋白存在相互作用,IspD与其相互作用最强的DXR蛋白之间通过氢键、盐桥等实现连接和相互作用。试验成功构建了表达菌株pET-32a-IspD,SDS-PAGE结果显示,在上清中高表达IspD融合蛋白;Western blotting验证显示,纯化的IspD融合蛋白具有反应原性。【结论】IspD蛋白具有成为候选诊断、疫苗研发和药物靶点等的潜在价值。试验表达纯化得到IspD融合蛋白,可为后续IspD蛋白具体功能的研究提供理论依据。

关键词: 单增李斯特菌; IspD蛋白; 生物信息学; 表达; 纯化

Abstract: 【Objective】 The aim of this study was to predict and analyze the potential biological function of IspD protein in Listeria monocytogenes,and express and purify IspD protein,which provided a theoretical support for subsequent research of IspD protein.【Method】 The coding sequence (gene ID:986270) and amino acid sequence (accession No.:NP_464611.1) of IspD protein in Listeria monocytogenes were obtained from NCBI.The bioinformatics software was used to analyze the open reading frame (ORF) of coding sequence of IspD protein,and predict and analyze the amino acid for basic characteristics,spatial structure,antigenic epitopes,modification sites,protein interaction and similarity.The coding sequence of IspD protein was codon optimized and synthesized.The recombinant plasmid pET-32a-IspD was constructed by seamless cloning method,then transformed into E.coli BL21(DE3) competent cells.Verified to be the correct positive clonal strain was confirmed by sequencing and double enzyme digestion was induced by IPTG overnight.The IspD fusion protein was purified by Ni-NTA spin columns.SDS-PAGE and Western blotting were used to verify the expression,purification and reactogenicity of IspD fusion protein.【Result】 The coding gene of IspD protein was lmo1086,the full-length was 711 bp,which had 4 ORFs,ORF1 was the longest ORF,which could encode 236 amino acids.Amino acid sequence analysis results showed that IspD was a hydrophobic protein without transmembrane domain and signal peptide,which was subcellular located in cytoplasm and was 2-C-methyl-D-ethrolitol-4-phosphate cytidylytransferase.Alpha helix,extended chain,beta turn and random coil were involved in the composition of the secondary structure,the prediction of the tertiary structure model was consistent with the characteristics of the secondary structure.IspD protein had multiple T cells and B cells epitopes,intrinsically disordered domain,phosphorylation,glycosylation,methylation and acetylation modification sites.There was a lot of protein interaction with IspD,such as DXR,IspF,ipk and so on,and IspD connected with DXR protein which was the strongest interaction through the hydrogen bonds and salt bridges.The expression strain pET-32a-IspD was successfully constructed.SDS-PAGE results showed that IspD fusion protein was highly expressed in the supernatant.Western blotting analysis indicated the purified IspD fusion protein had reactogenicity.【Conclusion】 IspD protein protein had potential value as a candidate diagnosis,vaccine development and drug target.IspD fusion protein was obtained by expression and purification,which provided a theoretical basis for subsequent study of the specific function of IspD protein.

Key words: Listeria monocytogenes; IspD protein; bioinformatics; expression; purification

中图分类号:

S852.61⁺5

史超, 张伟, 刘素平, 张梦琦, 何欣, 赵明彦, 周霞, 王震, 张辉. 单增李斯特菌IspD蛋白生物信息学分析及表达纯化[J]. 中国畜牧兽医, 2023, 50(9): 3799-3810.

SHI Chao, ZHANG Wei, LIU Suping, ZHANG Mengqi, HE Xin, ZHAO Mingyan, ZHOU Xia, WANG Zhen, ZHANG Hui. Bioinformatics Analysis and Expression Purification of IspD Protein in Listeria monocytogenes[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3799-3810.

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